Novel nucleic acid sequences encoding adenylate kinase, phospholipid scramblase-like, DNA fragmentation factor-like, phosphatidylserine synthase-like, and ATPase-like molecules and uses therefor

ABSTRACT

The invention provides isolated nucleic acids molecules that encode novel polypeptides. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing the nucleic acid molecules of the invention, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a sequence of the invention has been introduced or disrupted. The invention still further provides isolated proteins, fusion proteins, antigenic peptides and antibodies. Diagnostic methods utilizing compositions of the invention are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of 10/165,800, filed Jun. 7, 2002,which is a continuation-in-part of 09/781,677, filed Feb. 12, 2001,which claims the benefit of U.S. Provisional Application No. 60/181,705,filed Feb. 10, 2000, now abandoned; and a continuation-in-part of09/795,038, filed Feb. 26, 2001, now abandoned, which claims the benefitof U.S. Provisional Application No. 60/186,234, filed Feb. 29, 2000, nowabandoned; and a continuation-in-part of 09/790,180, filed Feb. 21,2001, now abandoned, which claims the benefit of U.S. ProvisionalApplication No. 60/185,947, filed Feb. 29, 2000, now abandoned; and acontinuation-in-part of 09/790,838, filed Feb. 22, 2001, now U.S. Pat.No. 6,489,152, which claims the benefit of U.S. Provisional ApplicationNo. 60/185,946, filed Feb. 29, 2000, now abandoned; and acontinuation-in-part of 09/790,179, filed Feb. 21, 2001, now U.S. Pat.No. 6,479,268, which claims the benefit of U.S. Provisional 60/185,609,filed Feb. 29, 2000, now abandoned; all of which are hereby incorporatedherein in their entirety by reference.

FIELD OF THE INVENTION

The invention relates to novel nucleic acid sequences and polypeptides.Also provided are vectors, host cells, and recombinant methods formaking and using the novel molecules.

TABLE OF CONTENTS

-   Chapter 1 7970, Novel ATPase-Like Molecule and Uses Thereof    -   i) SEQ ID NOS:1-4    -   ii) FIGS. 1-12    -   iii) Continuation-In-Part of Ser. No. 09/790,179, filed Feb. 21,        2001, which claims the benefit of U.S. Provisional 60/185,609,        filed Feb. 29, 2000-   Chapter 2 32670, Novel Human Phosphatidylserine Synthase-Like    Molecules and Uses Thereof    -   i) SEQ ID NOS: 5-9    -   ii) FIGS. 13-15    -   iii) Continuation-In-Part of Ser. No. 09/790,838, filed Feb. 22,        2001, which claims the benefit of U.S. Provisional 60/185,946,        filed Feb. 29, 2000-   Chapter 3 5698, A DNA Fragmentation Factor-Like Molecule and Uses    Thereof    -   i) SEQ ID NOS: 10-15    -   ii) FIGS. 16-20B    -   iii) Continuation-In-Part of Ser. No. 09/790,180, filed Feb. 21,        2001, which claims the benefit of U.S. Provisional Application        No. 60/185,947, filed Feb. 29, 2000-   Chapter 4 32621, Novel Human Phospholipid Scramblase-Like Molecules    and Uses Thereof    -   i) SEQ ID NOS: 16-20    -   ii) FIGS. 21-27    -   iii) Continuation-In-Part of Ser. No. 09/795,038, filed Feb. 26,        2001, which claims the benefit of U.S. Provisional Application        No. 60/186,234, filed Feb. 29, 2000-   Chapter 5 27802, A Novel Adenylate Kinase    -   i) SEQ ID NOS: 21-25    -   ii) FIGS. 28-35B    -   iii) Continuation-In-Part of 09/781, 677 filed Feb. 12, 2001,        which claims the benefit of U.S. Provisional Application No.        60/181,705, filed Feb. 10, 2000

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a hydropathy plot of a human ATPase-like molecule.Relative hydrophobic residues are shown above the dashed horizontalline, and relative hydrophilic residues are below the dashed horizontalline. The cysteine residues (cys) and N glycosylation site (Ngly) areindicated by short vertical lines just below the hydropathy trace. Thenumbers corresponding to the amino acid sequence (shown in SEQ ID NO:2)of human ATPase-like sequence are indicated. Polypeptides of theinvention include fragments which include: all or a part of ahydrophobic sequence (a sequence above the dashed line); or all or partof a hydrophilic fragment (a sequence below the dashed line). Otherfragments include a cysteine residue or as N-glycosylation site.

FIG. 2 depicts an alignment of the AAA (ATPases Associated to a varietyof cellular Activities) domain of the human ATPase-like sequence of theinvention with a consensus amino acid sequence derived from a hiddenMarkov model. The upper sequence is the consensus amino acid sequence(SEQ ID NO:4), while the lower amino acid sequence corresponds to aminoacids 128 to 312 of SEQ ID NO:2.

FIG. 3 shows an analysis of the 7970 amino acid sequence:αβ turn andcoil regions; hydrophilicity; amphipathic regions; flexible regions;antigenic index; and surface probability plot.

FIGS. 4A-B show the expression level of the 7970 mRNA transcript invarious tissues and cell lines.

FIG. 5 shows the expression level of the 7970 mRNA transcript in variousnormal and tumorous tissues and cell lines.

FIGS. 6A-B show the expression level of the 7970 mRNA transcript invarious normal and tumorous tissues.

FIG. 7 shows the expression level of the 7970 mRNA transcript inclinical angiogenic samples.

FIG. 8 shows the expression level of the 7970 transcript in clinicalcolon and liver samples.

FIG. 9 shows the expression level of the 7970 transcript in a non-smallcell lung cancer cell line (H640) in the presence and absence of the p16gene.

FIG. 10 shows the expression of the 7970 transcript in clinical breastsamples.

FIG. 11 shows the expression of the 7970 transcript in clinical ovarysamples.

FIG. 12 shows the expression of the 7970 transcript in clinical lungsamples.

FIG. 13 shows the amino acid sequence alignment for the protein (32670;SEQ ID NO:6) encoded by human 32670 (SEQ ID NO:5) with thephosphatidylserine synthase II from Cricetulus griseus (GI 2190007; NCBIAccession No. BAA20355; SEQ ID NO:8) and the phosphatidylserinesynthase-2 from Mus musculus (GI 4063700; NCBI Accession NumberAAC98383; SEQ ID NO:9). The sequence alignment was generated using theClustal method. The 32670 protein shares approximately 85% identity withthe phosphatidylserine synthase II from Cricetulus griseus andapproximately 86% identity with the murine phosphatidylserine synthase-2as determined by pairwise alignment.

FIGS. 14A-B provide the nucleotide and amino acid sequence (SEQ ID NO:5and 6, respectively) for clone 32670. The coding sequence for 32670 isshown in SEQ ID NO:7.

FIG. 15 shows a hydropathy plot of the 32670 polypeptide shown in SEQ IDNO:6. Relative hydrophobic residues are shown above the dashedhorizontal line, and relative hydrophilic residues are below the dashedhorizontal line. The cysteine residues (cys) and N glycosylation site(Ngly) are indicated by short vertical lines just below the hydropathytrace. The numbers corresponding to the amino acid sequence (shown inSEQ ID NO:6) of human 32670 are indicated. Polypeptides of the inventioninclude fragments which include: all or a part of a hydrophobic sequence(a sequence above the dashed line); or all or part of a hydrophilicfragment (a sequence below the dashed line). Other fragments include acysteine residue or as N-glycosylation site.

FIG. 16 depicts a hydropathy plot of human a DFF-like molecule. Relativehydrophobic residues are shown above the dashed horizontal line, andrelative hydrophilic residues are below the dashed horizontal line. Thecysteine residues (cys) and N glycosylation site (Ngly) are indicated byshort vertical lines just below the hydropathy trace. The numberscorresponding to the amino acid sequence (shown in SEQ ID NO:11) ofhuman DFF-like molecule are indicated. Polypeptides of the inventioninclude fragments which include: all or a part of a hydrophobic sequence(a sequence above the dashed line); or all or part of a hydrophilicfragment (a sequence below the dashed line). Other fragments include acysteine residue or as N-glycosylation site.

FIG. 17 depicts an alignment of the CAD domain of the human DFF-likemolecule with a consensus amino acid sequence derived from a hiddenMarkov model. The upper sequence is the consensus amino acid sequence(SEQ ID NO:13), while the lower amino acid sequence corresponds to aminoacids 36 to 108 of SEQ ID NO:11.

FIG. 18 shows an analysis of the 5698 amino acid sequence: αβturn andcoil regions; hydrophilicity; amphipathic regions; flexible regions;antigenic index; and surface probability plot.

FIG. 19 shows the amino acid sequence alignment for the protein (5698;SEQ ID NO:11) encoded by human 5698 (SEQ ID NO:10 or 12) with the Musmusculus cell death activator CIDE-B (SP Accession No. 3114594; GenbankAccession Number AAC34986; SEQ ID NO:14) and with the Homo sapiens celldeath activator CIDE-A (SP Accession No. 3114596; Genbank Accession No.AAC34987; SEQ ID NO:15). The sequence alignment was generated using theClustal method. The 5698 protein shares approximately 83% identity withthe murine CIDE-B and approximately 40% identity with the human CIDE-Aamino acid sequence as determined by pairwise alignment.

FIGS. 20A-B show the expression level of the 5698 mRNA transcript invarious normal and diseased human tissues.

FIG. 21 shows the amino acid sequence alignment for the protein (32621;SEQ ID NO:17) encoded by human 32621 (SEQ ID NO:16) with the murinephospholipid scramblase-like (SP Accession No. 2935163; GenbankAccession No. AAC40053; SEQ ID NO:19), and human Mm-1 cell derivedtransplantability-associated gene 1b (hMmTRA1b; SP Accession No.3510297; Genbank Accession No. BAA32568; SEQ ID NO:20). The sequencealignment was generated using the Clustal method. The 32621 proteinshares approximately 45% identity to the Mus musculus phospholipidscramblase-like and approximately 41% identity to the Homo sapienshMmTRA1b protein as determined by pairwise alignment.

FIGS. 22A-B provide the nucleotide and amino acid sequence (SEQ ID NO:16and 17, respectively) for clone 32621.

FIG. 23 depicts a hydropathy plot of human 32621. Relative hydrophobicresidues are shown above the dashed horizontal line, and relativehydrophilic residues are below the dashed horizontal line. The cysteineresidues (cys) and N glycosylation site (Ngly) are indicated by shortvertical lines just below the hydropathy trace. The numberscorresponding to the amino acid sequence (shown in SEQ ID NO:17) ofhuman 32621 are indicated. Polypeptides of the invention includefragments which include: all or a part of a hydrophobic sequence (asequence above the dashed line); or all or part of a hydrophilicfragment (a sequence below the dashed line). Other fragments include acysteine residue or as N-glycosylation site.

FIGS. 24A-B depict relative expression levels of 32621 in various humantissues and cells: artery (column 1); vein (column 2); aortic SMC,smooth muscle cells, early (column 3); aortic SMC late (column 4);static HUVEC, human umbilical vein endothelial cells (column 5); shearHUVEC (column 6); heart (column 7); heart CHF, congestive heart failureheart tissue (column 8); kidney (column 9); skeletal muscle (column 10);adipose (column 11); pancreas (column 12); primary osteoblasts (column13); osteoclasts (column 14); skin (column 15); spinal cord (column 16);brain cortex (column 17); brain hypothalamus (column 18); nerve (column19); DRG, dorsal root ganglion (column 20); glial cells (column 21);glioblastoma (column 22); breast (column 23); breast tumor (column 24);ovary (column 25); ovarian tumor (column 26); prostate (column 27);prostate tumor (column 28); prostate epithelial cells (column 29); colon(column 30); colon tumor (column 31); lung (column 32); lung tumor(column 33); lung COPD, chronic obstructive pulmonary diseased lung(column 34); colon IBD, inflammatory bowel diseased colon (column 35);liver (column 36); liver fibrosis (column 37); dermal cells (column 38);spleen (column 39); tonsil (column 40); lymph node (column 41); thymus(column 42); skin-decubitis (column 43); synovium (column 44); bonemarrow mononuclear cells (column 45); and activated peripheral bloodmononuclear cells (column 46). Expression levels were determined byquantitative RT-PCR (Taqman® brand quantitative PCR kit, AppliedBiosystems). The quantitative RT-PCR reactions were performed accordingto the kit manufacturer's instructions.

FIGS. 25A-B depict relative expression levels of 32621 in variousorgans: conf HMVEC, human microvascular endothelial cells (column 1);human fetal heart (column 2); human normal atrium (column 3); humannormal atrium (column 4); human normal ventricle (column 5); humannormal ventricle (column 6); human normal ventricle (column 7); humannormal ventricle (column 8); human normal ventricle (column 9); humanheart diseased ventricle (column 10); human heart diseased ventricle(column 11); human heart diseased ventricle (column 12); normal humankidney (column 13); normal human kidney (column 14); normal human kidney(column 15); normal human kidney (column 16); human kidney HT (column17); human kidney HT (column 18); human kidney HT (column 19); humankidney HT (column 20); human skeletal muscle (column 21); human skeletalmuscle (column 22); human liver (column 23); human liver withinflammation (column 24); fetal adrenal (column 25); Wilms Tumor (column26); Wilms Tumor (column 27); normal human spinal cord (column 28);diseased human cartilage (column 29); normal mouse atrium (column 30);normal mouse atrium (column 31); normal mouse ventricle (column 32); andnormal mouse ventricle (column 33). Relative expression levels weredetermined as described in FIGS. 24A-B.

FIG. 26 depicts relative expression levels of 32621 in various organ andliver samples including liver samples from animals fed modified diets:normal human heart (column 1); normal human kidney (column 2); normalhuman skeletal muscle (column 3); normal human liver (column 4); normalhuman liver (column 5); normal human liver (column 6); normal humanliver (column 7); normal human liver (column 8); normal human liver(column 9); normal human liver (column 10); diseased human liver (column11); diseased human liver (column 12); diseased human liver (column 13);diseased human liver (column 14); MK liver (chow diet) (column 15); MKliver (poly diet without chol., cholesterol) (column 16); MK liver (polydiet with chol.) (column 17); MK liver (chow diet) (column 18); MK liver(Sat. Diet without chol.) (column 19); and MK liver (Sat diet withchol.) (column 20). Relative expression levels were determined asdescribed in FIGS. 24A-B.

FIG. 27 depicts 32621 expression in various cell types: aortic smoothmuscle cells (ASMC)-A1PO, (column 1); ASMC-A2P3 (column 2); ASMC-A3P4(column 3); ASMC-AL (column 4); coronary artery smooth muscle cells(CASMC)-C1P3 (column 5); CASMC-C2P3 (column 6); CASMC-C5PO (column 7);CASMC-C1P6 (column 8); macrophage cells (column 9); macrophage cellstreated with interferon γ (column 10); CD40+ macrophage cells (column11); macrophage cells treated with lipopolysaccharide (column 12);HMVEC, human umbilical vein endothelial cells (column 13); HMVEC, humanmicrovascular endothelial cells (column 14); HAEC1, human aorticendothelial cells (column 15); HCAEC3, human coronary arterialendothelial cells (column 16); HCRE (column 17); RPTE, renal proximaltubule epithelial cells (column 18); MC (column 19); SKM1, myelogenousleukemia cells (column 20); and HLF, hepatocellular carcinoma cell line(column 21). Relative expression levels were determined as described inFIGS. 24A-B.

FIG. 28 shows the 27802 nucleotide sequence (SEQ ID NO:21) and thededuced amino acid sequence (SEQ ID NO:22).

FIG. 29 shows an analysis of the 27802 amino acid sequence: αβ turn andcoil regions; hydrophilicity; amphipathic regions; flexible regions;antigenic index; and surface probability plot.

FIG. 30 shows a 27802 receptor hydrophobicity plot. Relative hydrophobicresidues are shown above the dashed horizontal line, and relativehydrophilic residues are below the dashed horizontal line. The cysteineresidues (cys) and N glycosylation site (Ngly) are indicated by shortvertical lines just below the hydropathy trace. The numberscorresponding to the amino acid sequence (shown in SEQ ID NO:22) ofhuman 27802 are indicated. Polypeptides of the invention includefragments which include: all or a part of a hydrophobic sequence (asequence above the dashed line); or all or part of a hydrophilicfragment (a sequence below the dashed line). Other fragments include acysteine residue or an N-glycosylation site.

FIG. 31 shows an analysis of the 27802 open reading frame for aminoacids corresponding to specific functional sites. These sites arerelevant with regard to providing fragments of the 27802 nucleic acid orpeptide as disclosed herein.

FIG. 32 shows PSORT prediction of protein localization showing a highscore in the cytoplasm and significant scores in other cellularlocations.

FIG. 33 shows a description of ProDom matches for the 27802 protein.

FIG. 34 depicts an alignment of the adenylate kinase domains of human27802 with two consensus amino acid sequences derived from hidden Markovmodels. The upper sequence for domain 1 is the consensus amino acidsequence (SEQ ID NO:24) and the lower amino acid sequence corresponds toamino acids 41-120 of SEQ ID NO:22. The upper sequence for domain 2 isthe consensus amino acid sequence (SEQ ID NO:25) and the lower aminoacid sequence corresponds to amino acids 201-251 of SEQ ID NO:22.

FIGS. 35A-B display the expression levels of 27802 in various tissuesdetermined by quantitative PCR. The highest levels of expression of27802 were observed in artery, kidney, brain cortex and brainhypothalamus, ovary, lung (tumor), and tonsil. The tissue types are asfollows from left to right: Artery Normal, Aorta Diseased, Vein Normal,Coronary SMC, HUVEC, Hemangioma, Heart Normal, Heart CHF, Kidney,Skeletal Muscle, Adipose Normal, Pancreas, Primary Osteoblasts,Osteoclasts (diff), Skin Normal, Spinal Cord Normal, Brain CortexNormal, Brain Hypothalamus Normal, Nerve, DRG (Dorsal Root Ganglion),Breast Normal, Breast Tumor, Ovary Normal, Ovary Tumor, Prostate Normal,Prostate Tumor, Salivary Glands, Colon Normal, Colon Tumor, Lung Normal,Lung Tumor, Lung COPD, Colon IBD, Liver Normal, Liver Fibrosis, SpleenNormal, Tonsil Normal, Lymph Node Normal, Small Intestine Normal,Macrophages, Synovium, BM-MNC, Activated PBMC, Neutrophils,Megakaryocytes, Erythroid, Positive Control.

Chapter 1 7970, A Novel ATPase-Like Molecule and Uses Thereof BACKGROUNDOF THE INVENTION

Enzymes that bind to and hydrolyze ATP play a pivotal role intranslating chemically stored energy into biological activity. ATPasescan function in a variety of cellular processes including, selective iontransport events, actin-based motility, membrane traffic and numerousbiosynthetic pathways. Multiple ATPase families exist, including ionpumps, DEAD box-helicases, ABC transporters, and AAA (ATPases Associatedto a variety of cellular Activities).

AAA proteins play essential roles in cellular housekeeping, celldivision and differentiation and have been identified in prokaryotes andeukaryotes. All members of the AAA family are Mg²⁺ dependent ATPases andcomprise a conserved region that binds ATP. Cytosolic, transmembrane, aswell as, membrane-associated AAA family members have been identified invarious cellular locations and multimeric states.

The biological role of the AAA family members in the cell is diverse.Currently, members of this ATPase family are known to be involved inorganelle biogenesis, cell-cycle regulation, vesicle-mediated transport,assembly of proteins through membranes, peroxisome biogenesis, geneexpression in yeast and in human, and 26S proteasome function. For areview, see, Confalonieri et al. (1995) BioEssays 17:639-650.

The SEC18 gene product from S. cerevisiae is an AAA family member thatinfluences the transport of proteins between the endoplasmic reticulumand the golgi complex. It has been shown that SEC18 is an essentialcomponent of a multisubunit 20S “fusion machine” that promotes membranebilayer fusion coupled to ATP hydrolysis. The 20S fusion machine hasbeen proposed to be involved in the assembly, fusion or division of avariety of other membrane-bound subcellular compartments such asvacuoles, nuclei, mitochondria, or peroxisomes (Wilson et al. (1992) J.Cell. Bio. 117:531-538). Other AAA family members are involved inmitochondrial function. YME1 is a putative ATP and zinc-dependentprotease. Its inactivation leads to several morphological and functionaldefects, such as the escape of DNA from mitochondria (Thorsness et al.(1993) Mol Cell Biol 13: 5418-5426).

MSP1 is another AAA ATPase protein family member from yeast thatinfluences mitochondrial function. MSP1 is an intrinsic mitochondrialouter membrane protein with an apparent molecular mass of 40 KDa. MSP1is known to influence intramitochondrial protein sorting. Nakai et al.have demonstrated that the 61 mC1 fusion protein, normally localized tothe outer mitochondrial membrane, is mislocalized to the inner membraneof the mitochondria upon overexpression of MSP1 in yeast cell (Nakai etal. (1993) J. Biol. Chem. 268:24262-9).

Several members of the AAA family are involved in the biogenesis ofperoxisomes. These organelles contain enzymes responsible for fatty acidoxidation and the elimination of peroxides. AAA family members, such asthe PAS genes of S. cerevisiae, appear to be required for peroxisomegrowth, and proliferation (Subramani et al. (1993) Annu. Rev. Cell Biol.9:445-478). Furthemmore, mutations in the AAA proteins Pex1p or Pex6paccumulate abnormal peroxisomal vesicles, suggesting a defect in vesiclefusion during peroxisome assembly (Song et al. (1993) J. Cell Biol.123:535-548 and Heyman et al. (1994) J. Cell Biol. 127:1269-1273).

AAA family members are also known to regulate transcription. Nelbock etal. described the TBP1 protein that binds human HIV TAT transactivator,thus impairing its activity in cotransfection experiments (Nelbock etal. (1990) Science 248: 1650-1653). TBP1 has since been identified as anAAA family member which acts as a transcriptional activator for variouspromoters (Ohana et al. (1993) Proc. Natl. Acad. Sci. 90:138-142).

Various ATP-dependent protease, such as the regulatory components Lonand Clp, are also members of the AAA ATPase family. Evidence suggeststhe Lon and Clp proteases are involved in DNA replication, recombinationand restriction. For instance, human Lon binds specifically tosingle-stranded DNA in a region of the mitochondrial genome involved inregulation of DNA replication and transcription. It has been suggestedthat Lon may target and remodel specific DNA binding proteins either forselective degradation or for assembly (Fu et al. (1998) Biochemistry37:1905-1909).

Dubiel et al. discovered that subunit 4 of the human proteasome was infact a member of the AAA family (Dubiel et al. (1992) J. Biol. Chem.267:22699-22702). Subsequently, at least 5 of the 26S-proteasomesubunits already described as transcription factors or cell cycleproteins have now been identified as representatives of the AAA family.Therefore, members of the family are likely to play an essential role inATP-dependent and ubiquitin-dependent degradation of abnormal proteinsand short-lived regulatory proteins and in antigen processing.

Macromolecular machines (protein complexes) carry out nearly every majorprocess in a cell with highly coordinated moving parts driven by energydependent conformational changes. Examples of such structures includethe proteasomes, spliceosomes, ribosomes, peroxisomes and chromosomalreplicases. The intricacy of these machines require additional devicesto assist in their assembly. The AAA family of ATPase is thought of as aclass of molecular chaperones that assist in the noncovalent assembly ofother proteins or protein complexes. Thus, the AAA family members playcritical regulatory roles in the assembly or regulation of variousmolecular machines associated with diverse cellular activities.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize novel ATPases. The present inventionadvances the state of the art by providing a novel human ATPase-likenucleic acid and polypeptide.

SUMMARY OF THE INVENTION

Isolated nucleic acid molecules corresponding to ATPase-like nucleicacid sequences are provided. Additionally, amino acid sequencescorresponding to the polynucleotides are encompassed. In particular, thepresent invention provides for isolated nucleic acid moleculescomprising nucleotide sequences encoding the amino acid sequences shownin SEQ ID NO:2. Further provided are ATPase-like polypeptides having anamino acid sequence encoded by a nucleic acid molecule described herein.

The present invention also provides vectors and host cells forrecombinant expression of the nucleic acid molecules described herein,as well as methods of making such vectors and host cells and for usingthem for production of the polypeptides or peptides of the invention byrecombinant techniques.

The ATPase molecules of the present invention are useful for modulatingagents in a variety of cellular processes including organellebiogenesis, cell-cycle regulation, vesicle-mediated transport, assemblyof proteins through membranes, peroxisome biogenesis, protein sorting,gene expression, and 26S proteasome function. The molecules are alsouseful for the diagnosis and treatment of a variety of clinicalconditions.

Accordingly, in one aspect, this invention provides isolated nucleicacid molecules encoding ATPase-like proteins or biologically activeportions thereof, as well as nucleic acid fragments suitable as primersor hybridization probes for the detection of ATPase-like-encodingnucleic acids.

Another aspect of this invention features isolated or recombinantATPase-like proteins and polypeptides. Preferred ATPase-like proteinsand polypeptides possess at least one biological activity possessed bynaturally occurring ATPase proteins.

Variant nucleic acid molecules and polypeptides substantially homologousto the nucleotide and amino acid sequences set forth in the sequencelistings are encompassed by the present invention. Additionally,fragments and substantially homologous fragments of the nucleotide andamino acid sequences are provided.

Antibodies and antibody fragments that selectively bind the ATPase-likepolypeptides and fragments are provided. Such antibodies are useful indetecting the ATPase-like polypeptides as well as in regulating thecellular activities influenced by the ATPase-like polypeptide.

In another aspect, the present invention provides a method for detectingthe presence of ATPase-like activity or expression in a biologicalsample by contacting the biological sample with an agent capable ofdetecting an indicator of ATPase-like activity such that the presence ofATPase-like activity is detected in the biological sample.

In yet another aspect, the invention provides a method for modulatingATPase-like activity comprising contacting a cell with an agent thatmodulates (inhibits or stimulates) ATPase-like activity or expressionsuch that ATPase-like activity or expression in the cell is modulated.In one embodiment, the agent is an antibody that specifically binds toATPase-like proteins. In another embodiment, the agent modulatesexpression of ATPase-like protein by modulating transcription of anATPase-like gene, splicing of an ATPase-like mRNA, or translation of anATPase-like mRNA. In yet another embodiment, the agent is a nucleic acidmolecule having a nucleotide sequence that is antisense to the codingstrand of the ATPase-like mRNA or the ATPase-like gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant ATPase-likeprotein activity or nucleic acid expression by administering an agentthat is an ATPase-like modulator to the subject. In one embodiment, theATPase-like modulator is an ATPase-like protein. In another embodiment,the ATPase-like modulator is an ATPase-like nucleic acid molecule. Inother embodiments, the ATPase-like modulator is a peptide,peptidomimetic, or other small molecule.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic lesion or mutation characterized byat least one of the following: (1) aberrant modification or mutation ofa gene encoding an ATPase-like protein; (2) misregulation of a geneencoding an ATPase-like protein; and (3) aberrant post-translationalmodification of an ATPase-like protein, wherein a wild-type form of thegene encodes a protein with an ATPase-like activity.

In another aspect, the invention provides a method for identifying acompound that binds to or modulates the activity of an ATPase-likeprotein. In general, such methods entail measuring a biological activityof an ATPase-like protein in the presence and absence of a test compoundand identifying those compounds that alter the activity of theATPase-like protein.

The invention also features methods for identifying a compound thatmodulates the expression of ATPase-like genes by measuring theexpression of the ATPase-like sequences in the presence and absence ofthe compound.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the invention are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

The present invention provides ATPase-like molecules. By “ATPase-likemolecules” is intended a novel human sequence referred to as 7970, andvariants and fragments thereof. These full-length gene sequences orfragments thereof are referred to as “ATPase-like” sequences, indicatingthey share sequence similarity with ATPase genes. Isolated nucleic acidmolecules comprising nucleotide sequences encoding the 7970 polypeptidewhose amino acid sequence is given in SEQ ID NO:2, or a variant orfragment thereof, are provided. A nucleotide sequence encoding the 7970polypeptide is set forth in SEQ ID NO:1 and 3. The sequences are membersof the secretin family of ATPases.

A novel human ATPase-like gene sequence, referred to as 7970, isprovided. This gene sequence and variants and fragments thereof areencompassed by the term “ATPase-like” molecules or sequences as usedherein. The ATPase-like sequences find use in modulating a ATPasefunction. By “modulating” is intended the upregulating or downregulatingof a response. The sequences of the invention find use in modulatingorganelle biogenesis, cell-cycle regulation, protein degradation,vesicle-mediated transport, assembly of proteins through membranes,peroxisome biogenesis, gene expression, and 26S proteasome functionresponse. That is, the compositions of the invention, affect thetargeted activity in either a positive or negative fashion.

Proteins and/or antibodies of the invention are also useful inmodulating the above mentioned cellular process.

The present invention provides isolated nucleic acid moleculescomprising nucleotide sequences encoding the ATPase-like polypeptideswhose amino acid sequences are given in SEQ ID NO:2, or a variant orfragment of the polypeptides. Nucleotide sequences encoding theATPase-like polypeptides of the invention are set forth in SEQ ID NO:1and 3.

The disclosed invention relates to methods and compositions for themodulation, diagnosis, and treatment of a variety of disorders.Disorders involving the spleen include, but are not limited to,splenomegaly, including nonspecific acute splenitis, congestivespenomegaly, and spenic infarcts; neoplasms, congenital anomalies, andrupture. Disorders associated with splenomegaly include infections, suchas nonspecific splenitis, infectious mononucleosis, tuberculosis,typhoid fever, brucellosis, cytomegalovirus, syphilis, malaria,histoplasmosis, toxoplasmosis, kala-azar, trypanosomiasis,schistosomiasis, leishmaniasis, and echinococcosis; congestive statesrelated to partial hypertension, such as cirrhosis of the liver, portalor splenic vein thrombosis, and cardiac failure; lymphohematogenousdisorders, such as Hodgkin disease, non-Hodgkin lymphomas/leukemia,multiple myeloma, myeloproliferative disorders, hemolytic anemias, andthrombocytopenic purpura; immunologic-inflammatory conditions, such asrheumatoid arthritis and systemic lupus erythematosus; storage diseasessuch as Gaucher disease, Niemann-Pick disease, andmucopolysaccharidoses; and other conditions, such as amyloidosis,primary neoplasms and cysts, and secondary neoplasms.

Disorders involving the lung include, but are not limited to, congenitalanomalies; atelectasis; diseases of vascular origin, such as pulmonarycongestion and edema, including hemodynamic pulmonary edema and edemacaused by microvascular injury, adult respiratory distress syndrome(diffuse alveolar damage), pulmonary embolism, hemorrhage, andinfarction, and pulmonary hypertension and vascular sclerosis; chronicobstructive pulmonary disease, such as emphysema, chronic bronchitis,bronchial asthma, and bronchiectasis; diffuse interstitial(infiltrative, restrictive) diseases, such as pneumoconioses,sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitialpneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia(pulmonary infiltration with eosinophilia), Bronchiolitisobliterans—organizing pneumonia, diffuse pulmonary hemorrhage syndromes,including Goodpasture syndrome, idiopathic pulmonary hemosiderosis andother hemorrhagic syndromes, pulmonary involvement in collagen vasculardisorders, and pulmonary alveolar proteinosis; complications oftherapies, such as drug-induced lung disease, radiation-induced lungdisease, and lung transplantation; tumors, such as bronchogeniccarcinoma, including paraneoplastic syndromes, bronchioloalveolarcarcinoma, neuroendocrine tumors, such as bronchial carcinoid,miscellaneous tumors, and metastatic tumors; pathologies of the pleura,including inflammatory pleural effusions, noninflammatory pleuraleffusions, pneumothorax, and pleural tumors, including solitary fibroustumors (pleural fibroma) and malignant mesothelioma.

Disorders involving the colon include, but are not limited to,congenital anomalies, such as atresia and stenosis, Meckel diverticulum,congenital aganglionic megacolon-Hirschsprung disease; enterocolitis,such as diarrhea and dysentery, infectious enterocolitis, includingviral gastroenteritis, bacterial enterocolitis, necrotizingenterocolitis, antibiotic-associated colitis (pseudomembranous colitis),and collagenous and lymphocytic colitis, miscellaneous intestinalinflammatory disorders, including parasites and protozoa, acquiredimmunodeficiency syndrome, transplantation, drug-induced intestinalinjury, radiation enterocolitis, neutropenic colitis (typhlitis), anddiversion colitis; idiopathic inflammatory bowel disease, such as Crohndisease and ulcerative colitis; tumors of the colon, such asnon-neoplastic polyps, adenomas, familial syndromes, colorectalcarcinogenesis, colorectal carcinoma, and carcinoid tumors.

Disorders involving the liver include, but are not limited to, hepaticinjury; jaundice and cholestasis, such as bilirubin and bile formation;hepatic failure and cirrhosis, such as cirrhosis, portal hypertension,including ascites, portosystemic shunts, and splenomegaly; infectiousdisorders, such as viral hepatitis, including hepatitis A-E infectionand infection by other hepatitis viruses, clinicopathologic syndromes,such as the carrier state, asymptomatic infection, acute viralhepatitis, chronic viral hepatitis, and fulminant hepatitis; autoimmunehepatitis; drug- and toxin-induced liver disease, such as alcoholicliver disease; inborn errors of metabolism and pediatric liver disease,such as hemochromatosis, Wilson disease, α₁-antitrypsin deficiency, andneonatal hepatitis; intrahepatic biliary tract disease, such assecondary biliary cirrhosis, primary biliary cirrhosis, primarysclerosing cholangitis, and anomalies of the biliary tree; circulatorydisorders, such as impaired blood flow into the liver, including hepaticartery compromise and portal vein obstruction and thrombosis, impairedblood flow through the liver, including passive congestion andcentrilobular necrosis and peliosis hepatis, hepatic vein outflowobstruction, including hepatic vein thrombosis (Budd-Chiari syndrome)and veno-occlusive disease; hepatic disease associated with pregnancy,such as preeclampsia and eclampsia, acute fatty liver of pregnancy, andintrehepatic cholestasis of pregnancy; hepatic complications of organ orbone marrow transplantation, such as drug toxicity after bone marrowtransplantation, graft-versus-host disease and liver rejection, andnonimmunologic damage to liver allografts; tumors and tumorousconditions, such as nodular hyperplasias, adenomas, and malignanttumors, including primary carcinoma of the liver and metastatic tumors.

Disorders involving the uterus and endometrium include, but are notlimited to, endometrial histology in the menstrual cycle; functionalendometrial disorders, such as anovulatory cycle, inadequate lutealphase, oral contraceptives and induced endometrial changes, andmenopausal and postmenopausal changes; inflammations, such as chronicendometritis; adenomyosis; endometriosis; endometrial polyps;endometrial hyperplasia; malignant tumors, such as carcinoma of theendometrium; mixed Müllerian and mesenchymal tumors, such as malignantmixed Müllerian tumors; tumors of the myometrium, including leiomyomas,leiomyosarcomas, and endometrial stromal tumors.

Disorders involving the brain include, but are not limited to, disordersinvolving neurons, and disorders involving glia, such as astrocytes,oligodendrocytes, ependymal cells, and microglia; cerebral edema, raisedintracranial pressure and herniation, and hydrocephalus; malformationsand developmental diseases, such as neural tube defects, forebrainanomalies, posterior fossa anomalies, and syringomyelia and hydromyelia;perinatal brain injury; cerebrovascular diseases, such as those relatedto hypoxia, ischemia, and infarction, including hypotension,hypoperfusion, and low-flow states—global cerebral ischemia and focalcerebral ischemia—infarction from obstruction of local blood supply,intracranial hemorrhage, including intracerebral (intraparenchymal)hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, andvascular malformations, hypertensive cerebrovascular disease, includinglacunar infarcts, slit hemorrhages, and hypertensive encephalopathy;infections, such as acute meningitis, including acute pyogenic(bacterial) meningitis and acute aseptic (viral) meningitis, acute focalsuppurative infections, including brain abscess, subdural empyema, andextradural abscess, chronic bacterial meningoencephalitis, includingtuberculosis and mycobacterioses, neurosyphilis, and neuroborreliosis(Lyme disease), viral meningoencephalitis, including arthropod-borne(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes simplexvirus Type 2, Varicalla-zoster virus (Herpes zoster), cytomegalovirus,poliomyelitis, rabies, and human immunodeficiency virus 1, includingHIV-1 meningoencephalitis (subacute encephalitis), vacuolar myelopathy,AIDS-associated myopathy, peripheral neuropathy, and AIDS in children,progressive multifocal leukoencephalopathy, subacute sclerosingpanencephalitis, fungal meningoencephalitis, other infectious diseasesof the nervous system; transmissible spongiform encephalopathies (priondiseases); demyelinating diseases, including multiple sclerosis,multiple sclerosis variants, acute disseminated encephalomyelitis andacute necrotizing hemorrhagic encephalomyelitis, and other diseases withdemyelination; degenerative diseases, such as degenerative diseasesaffecting the cerebral cortex, including Alzheimer disease and Pickdisease, degenerative diseases of basal ganglia and brain stem,including Parkinsonism, idiopathic Parkinson disease (paralysisagitans), progressive supranuclear palsy, corticobasal degeneration,multiple system atrophy, including striatonigral degeneration,Shy-Drager syndrome, and olivopontocerebellar atrophy, and Huntingtondisease; spinocerebellar degenerations, including spinocerebellarataxias, including Friedreich ataxia, and ataxia-telanglectasia,degenerative diseases affecting motor neurons, including amyotrophiclateral sclerosis (motor neuron disease), bulbospinal atrophy (Kennedysyndrome), and spinal muscular atrophy; inborn errors of metabolism,such as leukodystrophies, including Krabbe disease, metachromaticleukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, andCanavan disease, mitochondrial encephalomyopathies, including Leighdisease and other mitochondrial encephalomyopathies; toxic and acquiredmetabolic diseases, including vitamin deficiencies such as thiamine(vitamin B₁) deficiency and vitamin B₁₂ deficiency, neurologic sequelaeof metabolic disturbances, including hypoglycemia, hyperglycemia, andhepatic encephatopathy, toxic disorders, including carbon monoxide,methanol, ethanol, and radiation, including combined methotrexate andradiation-induced injury; tumors, such as gliomas, includingastrocytoma, including fibrillary (diffuse) astrocytoma and glioblastomamultiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, andbrain stem glioma, oligodendroglioma, and ependymoma and relatedparaventricular mass lesions, neuronal tumors, poorly differentiatedneoplasms, including medulloblastoma, other parenchymal tumors,including primary brain lymphoma, germ cell tumors, and pinealparenchymal tumors, meningiomas, metastatic tumors, paraneoplasticsyndromes, peripheral nerve sheath tumors, including schwannoma,neurofibroma, and malignant peripheral nerve sheath tumor (malignantschwannoma), and neurocutaneous syndromes (phakomatoses), includingneurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindaudisease.

Disorders involving T-cells include, but are not limited to,cell-mediated hypersensitivity, such as delayed type hypersensitivityand T-cell-mediated cytotoxicity, and transplant rejection; autoimmunediseases, such as systemic lupus erythematosus, Sjögren syndrome,systemic sclerosis, inflammatory myopathies, mixed connective tissuedisease, and polyarteritis nodosa and other vasculitides; immunologicdeficiency syndromes, including but not limited to, primaryimmunodeficiencies, such as thymic hypoplasia, severe combinedimmunodeficiency diseases, and AIDS; leukopenia; reactive (inflammatory)proliferations of white cells, including but not limited to,leukocytosis, acute nonspecific lymphadenitis, and chronic nonspecificlymphadenitis; neoplastic proliferations of white cells, including butnot limited to lymphoid neoplasms, such as precursor T-cell neoplasms,such as acute lymphoblastic leukernia/lymphoma, peripheral T-cell andnatural killer cell neoplasms that include peripheral T-cell lymphoma,unspecified, adult T-cell leukemia/lymphoma, mycosis fungoides andSézary syndrome, and Hodgkin disease.

Diseases of the skin, include but are not limited to, disorders ofpigmentation and melanocytes, including but not limited to, vitiligo,freckle, melasma, lentigo, nevocellular nevus, dysplastic nevi, andmalignant melanoma; benign epithelial tumors, including but not limitedto, seborrheic keratoses, acanthosis nigricans, fibroepithelial polyp,epithelial cyst, keratoacanthoma, and adnexal (appendage) tumors;premalignant and malignant epidermal tumors, including but not limitedto, actinic keratosis, squamous cell carcinoma, basal cell carcinoma,and merkel cell carcinoma; tumors of the dermis, including but notlimited to, benign fibrous histiocytoma, dermatofibrosarcomaprotuberans, xanthomas, and dermal vascular tumors; tumors of cellularimmigrants to the skin, including but not limited to, histiocytosis X,mycosis fungoides (cutaneous T-cell lymphoma), and mastocytosis;disorders of epidermal maturation, including but not limited to,ichthyosis; acute inflammatory dermatoses, including but not limited to,urticaria, acute eczematous dermatitis, and erythema multiforme; chronicinflammatory dermatoses, including but not limited to, psoriasis, lichenplanus, and lupus erythematosus; blistering (bullous) diseases,including but not limited to, pemphigus, bullous pemphigoid, dermatitisherpetiformis, and noninflammatory blistering diseases: epidermolysisbullosa and porphyria; disorders of epidermal appendages, including butnot limited to, acne vulgaris; panniculitis, including but not limitedto, erythema nodosum and erythema induratum; and infection andinfestation, such as verrucae, molluscum contagiosum, impetigo,superficial fungal infections, and arthropod bites, stings, andinfestations.

In normal bone marrow, the myelocytic series (polymorphoneuclear cells)make up approximately 60% of the cellular elements, and the erythrocyticseries, 20-30%. Lymphocytes, monocytes, reticular cells, plasma cellsand megakaryocytes together constitute 10-20%. Lymphocytes make up 5-15%of normal adult marrow. In the bone marrow, cell types are add mixed sothat precursors of red blood cells (erythroblasts), macrophages(monoblasts), platelets (megakaryocytes), polymorphoneuclear leucocytes(myeloblasts), and lymphocytes (lymphoblasts) can be visible in onemicroscopic field. In addition, stem cells exist for the different celllineages, as well as a precursor stem cell for the committed progenitorcells of the different lineages. The various types of cells and stagesof each would be known to the person of ordinary skill in the art andare found, for example, on page 42 (FIG. 2-8) of Immunology,Imunopathology and Immunity, Fifth Edition, Sell et al. Simon andSchuster (1996), incorporated by reference for its teaching of celltypes found in the bone marrow. According, the invention is directed todisorders arising from these cells. These disorders include but are notlimited to the following: diseases involving hematopoeitic stem cells;committed lymphoid progenitor cells; lymphoid cells including B andT-cells; committed myeloid progenitors, including monocytes,granulocytes, and megakaryocytes; and committed erythroid progenitors.These include but are not limited to the leukemias, including B-lymphoidleukemias, T-lymphoid leukemias, undifferentiated leukemias;erythroleukemia, megakaryoblastic leukemia, monocytic; [leukemias areencompassed with and without differentiation; chronic and acutelymphoblastic leukemia, chronic and acute lymphocytic leukemia, chronicand acute myelogenous leukemia, lymphoma, myelo dysplastic syndrome,chronic and acute myeloid leukemia, myelomonocytic leukemia; chronic andacute myeloblastic leukemia, chronic and acute myelogenous leukemia,chronic and acute promyelocytic leukemia, chronic and acute myelocyticleukemia, hematologic malignancies of monocyte-macrophage lineage, suchas juvenile chronic myelogenous leukemia; secondary AML, antecedenthematological disorder; refractory anemia; aplastic anemia; reactivecutaneous angioendotheliomatosis; fibrosing disorders involving alteredexpression in dendritic cells, disorders including systemic sclerosis,E-M syndrome, epidemic toxic oil syndrome, eosinophilic fasciitislocalized forms of scleroderma, keloid, and fibrosing colonopathy;angiomatoid malignant fibrous histiocytoma; carcinoma, including primaryhead and neck squamous cell carcinoma; sarcoma, including kaposi'ssarcoma; fibroadanoma and phyllodes tumors, including mammaryfibroadenoma; stromal tumors; phyllodes tumors, including histiocytoma;erythroblastosis; neurofibromatosis; diseases of the vascularendothelium; demyelinating, particularly in old lesions; gliosis,vasogenic edema, vascular disease, Alzheimer's and Parkinson's disease;T-cell lymphomas; B-cell lymphomas.

Disorders involving the heart, include but are not limited to, heartfailure, including but not limited to, cardiac hypertrophy, left-sidedheart failure, and right-sided heart failure; ischemic heart disease,including but not limited to angina pectoris, myocardial infarction,chronic ischemic heart disease, and sudden cardiac death; hypertensiveheart disease, including but not limited to, systemic (left-sided)hypertensive heart disease and pulmonary (right-sided) hypertensiveheart disease; valvular heart disease, including but not limited to,valvular degeneration caused by calcification, such as calcific aorticstenosis, calcification of a congenitally bicuspid aortic valve, andmitral annular calcification, and myxomatous degeneration of the mitralvalve (mitral valve prolapse), rheumatic fever and rheumatic heartdisease, infective endocarditis, and noninfected vegetations, such asnonbacterial thrombotic endocarditis and endocarditis of systemic lupuserythematosus (Libman-Sacks disease), carcinoid heart disease, andcomplications of artificial valves; myocardial disease, including butnot limited to dilated cardiomyopathy, hypertrophic cardiomyopathy,restrictive cardiomyopathy, and myocarditis; pericardial disease,including but not limited to, pericardial effusion and hemopericardiumand pericarditis, including acute pericarditis and healed pericarditis,and rheumatoid heart disease; neoplastic heart disease, including butnot limited to, primary cardiac tumors, such as myxoma, lipoma,papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac effectsof noncardiac neoplasms; congenital heart disease, including but notlimited to, left-to-right shunts—late cyanosis, such as atrial septaldefect, ventricular septal defect, patent ductus arteriosus, andatrioventricular septal defect, right-to-left shunts—early cyanosis,such as tetralogy of fallot, transposition of great arteries, truncusarteriosus, tricuspid atresia, and total anomalous pulmonary venousconnection, obstructive congenital anomalies, such as coarctation ofaorta, pulmonary stenosis and atresia, and aortic stenosis and atresia,and disorders involving cardiac transplantation.

Disorders involving blood vessels include, but are not limited to,responses of vascular cell walls to injury, such as endothelialdysfunction and endothelial activation and intimal thickening; vasculardiseases including, but not limited to, congenital anomalies, such asarteriovenous fistula, atherosclerosis, and hypertensive vasculardisease, such as hypertension; inflammatory disease—the vasculitides,such as giant cell (temporal) arteritis, Takayasu arteritis,polyarteritis nodosa (classic), Kawasaki syndrome (mucocutaneous lymphnode syndrome), microscopic polyanglitis (microscopic polyarteritis,hypersensitivity or leukocytoclastic anglitis), Wegener granulomatosis,thromboanglitis obliterans (Buerger disease), vasculitis associated withother disorders, and infectious arteritis; Raynaud disease; aneurysmsand dissection, such as abdominal aortic aneurysms, syphilitic (luetic)aneurysms, and aortic dissection (dissecting hematoma); disorders ofveins and lymphatics, such as varicose veins, thrombophlebitis andphlebothrombosis, obstruction of superior vena cava (superior vena cavasyndrome), obstruction of inferior vena cava (inferior vena cavasyndrome), and lymphangitis and lymphedema; tumors, including benigntumors and tumor-like conditions, such as hemangioma, lymphangioma,glomus tumor (glomangioma), vascular ectasias, and bacillaryangiomatosis, and intermediate-grade (borderline low-grade malignant)tumors, such as Kaposi sarcoma and hemangloendothelioma, and malignanttumors, such as angiosarcoma and hemangiopericytoma; and pathology oftherapeutic interventions in vascular disease, such as balloonangioplasty and related techniques and vascular replacement, such ascoronary artery bypass graft surgery.

Disorders involving red cells include, but are not limited to, anemias,such as hemolytic anemias, including hereditary spherocytosis, hemolyticdisease due to erythrocyte enzyme defects: glucose-6-phosphatedehydrogenase deficiency, sickle cell disease, thalassemia syndromes,paroxysmal nocturnal hemoglobinuria, immunohemolytic anemia, andhemolytic anemia resulting from trauma to red cells; and anemias ofdiminished erythropoiesis, including megaloblastic anemias, such asanemias of vitamin B12 deficiency: pernicious anemia, and anemia offolate deficiency, iron deficiency anemia, anemia of chronic disease,aplastic anemia, pure red cell aplasia, and other forms of marrowfailure.

Disorders involving the thymus include developmental disorders, such asDiGeorge syndrome with thymic hypoplasia or aplasia; thymic cysts;thymic hypoplasia, which involves the appearance of lymphoid follicleswithin the thymus, creating thymic follicular hyperplasia; and thymomas,including germ cell tumors, lynphomas, Hodgkin disease, and carcinoids.Thymomas can include benign or encapsulated thymoma, and malignantthymoma Type I (invasive thymoma) or Type II, designated thymiccarcinoma.

Disorders involving B-cells include, but are not limited to precursorB-cell neoplasms, such as lymphoblastic leukemia/lymphoma. PeripheralB-cell neoplasms include, but are not limited to, chronic lymphocyticleukemia/small lymphocytic lymphoma, follicular lymphoma, diffuse largeB-cell lymphoma, Burkitt lymphoma, plasma cell neoplasms, multiplemyeloma, and related entities, lymphoplasmacytic lymphoma (Waldenstrommacroglobulinemia), mantle cell lymphoma, marginal zone lymphoma(MALToma), and hairy cell leukemia.

Disorders involving the kidney include, but are not limited to,congenital anomalies including, but not limited to, cystic diseases ofthe kidney, that include but are not limited to, cystic renal dysplasia,autosomal dominant (adult) polycystic kidney disease, autosomalrecessive (childhood) polycystic kidney disease, and cystic diseases ofrenal medulla, which include, but are not limited to, medullary spongekidney, and nephronophthisis-uremic medullary cystic disease complex,acquired (dialysis-associated) cystic disease, such as simple cysts;glomerular diseases including pathologies of glomerular injury thatinclude, but are not limited to, in situ immune complex deposition, thatincludes, but is not limited to, anti-GBM nephritis, Heymann nephritis,and antibodies against planted antigens, circulating immune complexnephritis, antibodies to glomerular cells, cell-mediated immunity inglomerulonephritis, activation of alternative complement pathway,epithelial cell injury, and pathologies involving mediators ofglomerular injury including cellular and soluble mediators, acuteglomerulonephritis, such as acute proliferative (poststreptococcal,postinfectious) glomerulonephritis, including but not limited to,poststreptococcal glomerulonephritis and nonstreptococcal acuteglomerulonephritis, rapidly progressive (crescentic) glomerulonephritis,nephrotic syndrome, membranous glomerulonephritis (membranousnephropathy), minimal change disease (lipoid nephrosis), focal segmentalglomeruloscierosis, membranoproliferative glomerulonephritis, IgAnephropathy (Berger disease), focal proliferative and necrotizingglomerulonephritis (focal glomerulonephritis), hereditary nephritis,including but not limited to, Alport syndrome and thin membrane disease(benign familial hematuria), chronic glomerulonephritis, glomerularlesions associated with systemic disease, including but not limited to,systemic lupus erythematosus, Henoch-Schonlein purpura, bacterialendocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary andimmunotactoid glomerulonephritis, and other systemic disorders; diseasesaffecting tubules and interstitium, including acute tubular necrosis andtubulointerstitial nephritis, including but not limited to,pyelonephritis and urinary tract infection, acute pyelonephritis,chronic pyelonephritis and reflux nephropathy, and tubulointerstitialnephritis induced by drugs and toxins, including but not limited to,acute drug-induced interstitial nephritis, analgesic abuse nephropathy,nephropathy associated with nonsteroidal anti-inflammatory drugs, andother tubulointerstitial diseases including, but not limited to, uratenephropathy, hypercalcemia and nephrocalcinosis, and multiple myeloma;diseases of blood vessels including benign nephrosclerosis, malignanthypertension and accelerated nephrosclerosis, renal artery stenosis, andthrombotic microangiopathies including, but not limited to, classic(childhood) hemolytic-uremic syndrome, adult hemolytic-uremicsyndrome/thrombotic thrombocytopenic purpura, idiopathic HUS/TTP, andother vascular disorders including, but not limited to, atheroscleroticischemic renal disease, atheroembolic renal disease, sickle cell diseasenephropathy, diffuse cortical necrosis, and renal infarcts; urinarytract obstruction (obstructive uropathy); urolithiasis (renal calculi,stones); and tumors of the kidney including, but not limited to, benigntumors, such as renal papillary adenoma, renal fibroma or hamartoma(renomedullary interstitial cell tumor), angiomyolipoma, and oncocytoma,and malignant tumors, including renal cell carcinoma (hypernephroma,adenocarcinoma of kidney), which includes urothelial carcinomas of renalpelvis.

Disorders of the breast include, but are not limited to, disorders ofdevelopment; inflammations, including but not limited to, acutemastitis, periductal mastitis, periductal mastitis (recurrent subareolarabscess, squamous metaplasia of lactiferous ducts), mammary ductectasia, fat necrosis, granulomatous mastitis, and pathologiesassociated with silicone breast implants; fibrocystic changes;proliferative breast disease including, but not limited to, epithelialhyperplasia, sclerosing adenosis, and small duct papillomas; tumorsincluding, but not limited to, stromal tumors such as fibroadenoma,phyllodes tumor, and sarcomas, and epithelial tumors such as large ductpapilloma; carcinoma of the breast including in situ (noninvasive)carcinoma that includes ductal carcinoma in situ (including Paget'sdisease) and lobular carcinoma in situ, and invasive (infiltrating)carcinoma including, but not limited to, invasive ductal carcinoma, nospecial type, invasive lobular carcinoma, medullary carcinoma, colloid(mucinous) carcinoma, tubular carcinoma, and invasive papillarycarcinoma, and miscellaneous malignant neoplasms.

Disorders in the male breast include, but are not limited to,gynecomastia and carcinoma.

Disorders involving the testis and epididymis include, but are notlimited to, congenital anomalies such as cryptorchidism, regressivechanges such as atrophy, inflammations such as nonspecific epididymitisand orchitis, granulomatous (autoimmune) orchitis, and specificinflammations including, but not limited to, gonorrhea, mumps,tuberculosis, and syphilis, vascular disturbances including torsion,testicular tumors including germ cell tumors that include, but are notlimited to, seminoma, spermatocytic seminoma, embryonal carcinoma, yolksac tumor choriocarcinoma, teratoma, and mixed tumors, tumore of sexcord-gonadal stroma including, but not limited to, Leydig (interstitial)cell tumors and sertoli cell tumors (androblastoma), and testicularlymphoma, and miscellaneous lesions of tunica vaginalis.

Disorders involving the prostate include, but are not limited to,inflammations, benign enlargement, for example, nodular hyperplasia(benign prostatic hypertrophy or hyperplasia), and tumors such ascarcinoma.

Disorders involving the thyroid include, but are not limited to,hyperthyroidism; hypothyroidism including, but not limited to, cretinismand myxedema; thyroiditis including, but not limited to, hashimotothyroiditis, subacute (granulomatous) thyroiditis, and subacutelymphocytic (painless) thyroiditis; Graves disease; diffuse andmultinodular goiter including, but not limited to, diffuse nontoxic(simple) goiter and multinodular goiter; neoplasms of the thyroidincluding, but not limited to, adenomas, other benign tumors, andcarcinomas, which include, but are not limited to, papillary carcinoma,follicular carcinoma, medullary carcinoma, and anaplastic carcinoma; andcogenital anomalies.

Disorders involving the skeletal muscle include tumors such asrhabdomyosarcoma.

Disorders involving the pancreas include those of the exocrine pancreassuch as congenital anomalies, including but not limited to, ectopicpancreas; pancreatitis, including but not limited to, acutepancreatitis; cysts, including but not limited to, pseudocysts; tumors,including but not limited to, cystic tumors and carcinoma of thepancreas; and disorders of the endocrine pancreas such as, diabetesmellitus; islet cell tumors, including but not limited to, insulinomas,gastrinomas, and other rare islet cell tumors.

Disorders involving the small intestine include the malabsorptionsyndromes such as, celiac sprue, tropical sprue (postinfectious sprue),whipple disease, disaccharidase (lactase) deficiency,abetalipoproteinemia, and tumors of the small intestine includingadenomas and adenocarcinoma.

Disorders related to reduced platelet number, thrombocytopenia, includeidiopathic thrombocytopenic purpura, including acute idiopathicthrombocytopenic purpura, drug-induced thrombocytopenia, HIV-associatedthrombocytopenia, and thrombotic microangiopathies: thromboticthrombocytopenic purpura and hemolytic-uremic syndrome.

Disorders involving precursor T-cell neoplasms include precursor Tlymphoblastic leukemia/lymphoma. Disorders involving peripheral T-celland natural killer cell neoplasms include T-cell chronic lymphocyticleukemia, large granular lymphocytic leukemia, mycosis fungoides andSezary syndrome, peripheral T-cell lymphoma, unspecified,angioimmunoblastic T-cell lymphoma, angiocentric lymphoma (NK/T-celllymphoma^(4a)), intestinal T-cell lymphoma, adult T-cellleukemia/lymphoma, and anaplastic large cell lymphoma.

Disorders involving the ovary include, for example, polycystic ovariandisease, Stein-leventhal syndrome, Pseudomyxoma peritonei and stromalhyperthecosis; ovarian tumors such as, tumors of coelomic epithelium,serous tumors, mucinous tumors, endometeriod tumors, clear celladenocarcinoma, cystadenofibroma, brenner tumor, surface epithelialtumors; germ cell tumors such as mature (benign) teratomas, monodermalteratomas, immature malignant teratomas, dysgerminoma, endodermal sinustumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-thecacell tumors, thecoma-fibromas, androblastomas, hill cell tumors, andgonadoblastoma; and metastatic tumors such as Krukenberg tumors.

Bone-forming cells include the osteoprogenitor cells, osteoblasts, andosteocytes. The disorders of the bone are complex because they may havean impact on the skeleton during any of its stages of development.Hence, the disorders may have variable manifestations and may involveone, multiple or all bones of the body. Such disorders include,congenital malformations, achondroplasia and thanatophoric dwarfism,diseases associated with abnormal matix such as type 1 collagen disease,osteoporosis, Paget disease, rickets, osteomalacia, high-turnoverosteodystrophy, low-turnover of aplastic disease, osteonecrosis,pyogenic osteomyelitis, tuberculous osteomyelitism, osteoma, osteoidosteoma, osteoblastoma, osteosarcoma, osteochondroma, chondromas,chondroblastoma, chondromyxoid fibroma, chondrosarcoma, fibrous corticaldefects, fibrous dysplasia, fibrosarcoma, malignant fibroushistiocytoma, Ewing sarcoma, primitive neuroectodermal tumor, giant celltumor, and metastatic tumors.

The ATPase-like gene, clone 7970, was identified in a cDNA library.Clone 7970 encodes a mRNA transcript having the corresponding cDNA setforth in SEQ ID NO:1. This transcript has a 1086 nucleotide open readingframe (nucleotides 79-1165 of SEQ ID NO:1), which encodes a 361 aminoacid protein (SEQ ID NO:2). An analysis of the full-length 7970polypeptide predicts that the N-terminal 41 amino acids represent asignal peptide. Transmembrane segments from amino acids (aa) 16-34 and173-191 were predicted by MEMSAT. Transmembrane segments were alsopredicted from aa 133-151 of the presumed mature peptide sequence.Prosite program analysis was used to predict various sites within the7970 protein. N-glycosylation sites were predicted at aa 200-203 and316-319. Protein kinase C phosphorylation sites were predicted at aa33-35, 44-46, 146-148, 163-165, 170-172 and 273-239. Casein kinase IIphosphorylation sites were predicted at aa 12-15, 70-73, 89-92, 146-149,161-164, 202-205, 218-212, 295-298, 317-320, and 322-325. AnN-myristoylation site is predicted at aa 136-141. An ATP/GTP-bindingsite motif A (P-loop) was predicted at aa 133-140. A Leucine zipperpattern was predicted at aa 109-130.

The ATPase-like protein possesses a NB-ARC domain, from aa 131-145, anAAA domain from aa 128-312, an adenylate kinase domain from aa 131-139,a RNA helicase domain from aa 7-337, as predicted by HMMer, Version 2.For general information regarding PFAM identifiers, PS prefix and PFprefix domain identification numbers, refer to Sonnhammer et al. (1997)Protein 28:405-420 andhttp//www.psc.edu/general/software/packages/pfam/pfam.html. The NB-ARCdomain is a novel signaling motif shared by plant resistant geneproducts and regulators of cell death in animals. See for example, Vander Biezen et al. (1998) Curr Biol 8:229-227. Adenylate kinase is asmall monomeric enzyme that catalyzes the reversible transfer of MgATPto AMP. In mammals there are three different isozymes: AK1 (ormyokinase), which is cytosolic; AK2, which is located in the outercompartment of mitochondria; and, AK3 (or GTP:AMP phosphotransferase),which is located in the mitochondrial matrix and which uses MgGTPinstead of MgATP. The RNA helices domain is found in a family of RNAhelices thought to be involved in duplex unwinding during viral RNAreplication. Members of this family are found in a variety of singlestranded RNA viruses. See for example, Gorbalenya et al. (1989) NAR17:4713-4730. The AAA domain (ATPase Associated with various cellularActivities) is found in a family of proteins that often performchaperone-like functions that assist in the assembly, operation, ordisassembly of protein complexes. See for example, Confalonieri et al.(1995) Bioessays 17:639-650 and Neuwald et al. (1999) Genome Research9:27-43.

The 7970 protein displays 37% identity from aa 202-312, 34% identityfrom aa 154-292, and 57% identity to aa 128-165 to a Prodom consensussequence found in members of the Lon family of ATP-dependent proteases;25% identity from aa 13-127 to a Prodom consensus sequence found in theMSP1 protein homolog; 25% identity from aa 126-336 to a Prodom consensussequence found in a putative cell division protein from Treponemapallidum; 22% identity from aa 130-347 to a Prodom consensus sequencefound in a probable Peroxin-6 protein which may play a role inbiogenesis of peroxisomes; 35% identity from aa 77-351 and 31% identityfrom aa 324-351 to a Prodom consensus sequence found in members of thepeptidase family 16S; 20% identity from aa 129-355 to a Prodom consensussequence found in a chromosomal replication initiator protein; and, 32%identity from aa 117-198 to a Prodom consensus sequence found in aTAT-binding homolog. Furthermore, the amino acid sequence of the 7970sequence shares approximately 47% sequence identity with the C. elegansMSPI protein homolog (Genbank Accession No. P54815). This sequencealignment was generated using the clustal method.

The ATPase-like sequences of the invention are members of a family ofmolecules (the “ATPase family”) having conserved functional features.The term “family” when referring to the proteins and nucleic acidmolecules of the invention is intended to mean two or more proteins ornucleic acid molecules having sufficient amino acid or nucleotidesequence identity as defined herein. Such family members can benaturally occurring and can be from either the same or differentspecies. For example, a family can contain a first protein of murineorigin and a homologue of that protein of human origin, as well as asecond, distinct protein of human origin and a murine homologue of thatprotein. Members of a family may also have common functionalcharacteristics.

As used herein, the term “AAA domain” includes an amino acid sequence ofabout 80-184 amino acid residues in length and having a bit score forthe alignment of the sequence to the AAA domain (HMM) of at least 8.Preferably, an AAA domain includes at least about 50-200 amino acids,more preferably about 100-185 amino acid residues, or about 81-185 aminoacids and has a bit score for the alignment of the sequence to the AAAdomain (HMM) of at least 16 or greater. The AAA domain (HMM) has beenassigned the PFAM Accession No. PF00004 (http://pfam.wustl.edu/). Analignment of the AAA domain (amino acids 128 to 312 of SEQ ID NO:2) ofthe human ATPase-like sequence with a consensus amino acid sequencederived from a hidden Markov model is depicted in FIG. 3.

In a preferred embodiment ATPase-like polypeptide or protein has a “AAAdomain” or a region which includes at least about 100-250 morepreferably about 130-200 or 160-200 amino acid residues and has at leastabout 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with an“AAA domain,” e.g., the AAA domain of human ATPase-like molecule (e.g.,amino acid residues 128-312 of SEQ ID NO:2).

To identify the presence of an “AAA” domain in an ATPase-like proteinsequence, and make the determination that a polypeptide or protein ofinterest has a particular profile, the amino acid sequence of theprotein can be searched against a database of HMMs (e.g., the Pfamdatabase, release 2.1) using the default parameters(http://www.sanger.ac.uk/Software/Pfam/HMM_search). For example, thehmmsf program, which is available as part of the HMMER package of searchprograms, is a family specific default program for MILPAT0063 and ascore of 15 is the default threshold score for determining a hit.Alternatively, the threshold score for determining a hit can be lowered(e.g., to 8 bits). A description of the Pfam database can be found inSonhammer et al. (1997) Proteins 28(3):405-420 and a detaileddescription of HMMs can be found, for example, in Gribskov et al. (1990)Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad.Sci. USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531;and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of whichare incorporated herein by reference.

In one embodiment, an ATPase-like protein includes at least onetransmembrane domain. As used herein, the term “transmembrane domain”includes an amino acid sequence of about 15 amino acid residues inlength that spans a phospholipid membrane. More preferably, atransmembrane domain includes about at least 18, 20, 22, 24, 25, 30,30.5 or 40 amino acid residues and spans a phospholipid membrane.Transmembrane domains are rich in hydrophobic residues, and typicallyhave an α-helical structure. In a preferred embodiment, at least 50%,60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembranedomain are hydrophobic, e.g., leucines, isoleucines, tyrosines, ortryptophans. Transmembrane domains are described in, for example,http://pfam.wustl.edu/cgi-bin/getdesc?name=7tm-1, and Zagotta W. N. etal. (1996) Annual Rev. Neuronsci. 19:235-63, the contents of which areincorporated herein by reference.

In a preferred embodiment, an ATPase-like] polypeptide or protein has atleast one transmembrane domain or a region which includes at least 18,20, 22, 24, 25, 30, 35 or 40 amino acid residues and has at least about60%, 70% 80% 90% 95%, 99%, or 100% sequence identity with a“transmembrane domain,” e.g., at least one transmembrane domain of humanATPase-like (e.g., amino acid residues 18 of SEQ ID NO:2).

In another embodiment, an ATPase-like protein includes at least one“non-transmembrane domain.” As used herein, “non-transmembrane domains”are domains that reside outside of the membrane. When referring toplasma membranes, non-transmembrane domains include extracellulardomains (i.e., outside of the cell) and intracellular domains (i.e.,within the cell). When referring to membrane-bound proteins found inintracellular organelles (e.g., mitochondria, endoplasmic reticulum,peroxisomes and microsomes), non-transmembrane domains include thosedomains of the protein that reside in the cytosol (i.e., the cytoplasm),the lumen of the organelle, or the matrix or the intermembrane space(the latter two relate specifically to mitochondria organelles). TheC-terminal amino acid residue of a non-transmembrane domain is adjacentto an N-terminal amino acid residue of a transmembrane domain in anaturally occurring ATPase-like, or ATPase-like protein.

In a preferred embodiment, an ATPase-like polypeptide or protein has a“non-transmembrane domain” or a region which includes at least about1-170, preferably about 100-170, about 50-160, and about 20-125 aminoacid residues, and has at least about 60%, 70% 80% 90% 95%, 99% or 100%sequence identity with a “non-transmembrane domain”, e.g., anon-transmembrane domain of human ATPase-like (e.g., residues 191 and361 of SEQ ID NO:2). Preferably, a non-transmembrane domain is capableof catalytic activity (e.g., ATPase-like activity).

A non-transmembrane domain located at the N-terminus of an ATPase-likeprotein or polypeptide is referred to herein as an “N-terminalnon-transmembrane domain.” As used herein, an “N-terminalnon-transmembrane domain” includes an amino acid sequence having about1-350, preferably about 30-325, more preferably about 50-320, or evenmore preferably about 80-310 amino acid residues in length and islocated outside the boundaries of a membrane. For example, an N-terminalnon-transmembrane domain is located at about amino acid residues 1-15 ofSEQ ID NO:2.

Similarly, a non-transmembrane domain located at the C-terminus of anATPase-like protein or polypeptide is referred to herein as a“C-terminal non-transmembrane domain.” As used herein, an “C-terminalnon-transmembrane domain” includes an amino acid sequence having about1-300, preferably about 15-290, preferably about 20-270, more preferablyabout 25-255 amino acid residues in length and is located outside theboundaries of a membrane. For example, an C-terminal non-transmembranedomain is located at about amino acid residues 191-361 of SEQ ID NO:2.

An ATPase-like molecule can further include a signal sequence. As usedherein, a “signal sequence” refers to a peptide of about 20-80 aminoacid residues in length which occurs at the N-terminus of secretory andintegral membrane proteins and which contains a majority of hydrophobicamino acid residues. For example, a signal sequence contains at leastabout 12-25 amino acid residues, preferably about 30-70 amino acidresidues, more preferably about 61 amino acid residues, and has at leastabout 40-70%, preferably about 50-65%, and more preferably about 55-60%hydrophobic amino acid residues (e.g., alanine, valine, leucine,isoleucine, phenylalanine, tyrosine, tryptophan, or proline). Such a“signal sequence”, also referred to in the art as a “signal peptide”,serves to direct a protein containing such a sequence to a lipidbilayer. For example, in one embodiment, an ATPase-like protein containsa signal sequence of about amino acids 1-41 of SEQ ID NO:2. The “signalsequence” is cleaved during processing of the mature protein. The matureATPase-like protein corresponds to amino acids 42-361 of SEQ ID NO:2.

Preferred ATPase-like polypeptides of the present invention have anamino acid sequence sufficiently identical to the amino acid sequence ofSEQ ID NO:2. The term “sufficiently identical” is used herein to referto a first amino acid or nucleotide sequence that contains a sufficientor minimum number of identical or equivalent (e.g., with a similar sidechain) amino acid residues or nucleotides to a second amino acid ornucleotide sequence such that the first and second amino acid ornucleotide sequences have a common structural domain and/or commonfunctional activity. For example, amino acid or nucleotide sequencesthat contain a common structural domain having at least about 45%, 55%,or 65% identity, preferably 75% identity, more preferably 85%, 95%, or98% identity are defined herein as sufficiently identical.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. The percent identity between two sequences can bedetermined using techniques similar to those described below, with orwithout allowing gaps. In calculating percent identity, typically exactmatches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. In a preferred embodiment,the percent identity between two amino acid sequences is determinedusing the Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453 algorithmwhich has been incorporated into the GAP program in the GCG softwarepackage (available at http://www.gcg.com), using either a Blossum 62matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferredembodiment, the percent identity between two nucleotide sequences isdetermined using the GAP program in the GCG software package (availableat http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused if the practitioner is uncertain about what parameters should beapplied to determine if a molecule is within a sequence identity orhomology limitation of the invention) is using a Blossum 62 scoringmatrix with a gap open penalty of 12, a gap extend penalty of 4, and aframeshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of Karlin and Altschul (1990) Proc.Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al.(1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performedwith the NBLAST program, score=100, wordlength=12, to obtain nucleotidesequences homologous to ATPase-like nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3, to obtain amino acid sequenceshomologous to ATPase-like protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-Blast can be used to perform an iterated search thatdetects distant relationships between molecules. See Altschul et al.(1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blastprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Anotherpreferred, non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Myers and Miller (1988)CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program(version 2.0), which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

Accordingly, another embodiment of the invention features isolatedATPase-like proteins and polypeptides having an ATPase-like proteinactivity. As used interchangeably herein, a “ATPase-like proteinactivity”, “biological activity of an ATPase-like protein”, or“functional activity of an ATPase-like protein” refers to an activityexerted by an ATPase-like protein, polypeptide, or nucleic acid moleculeon an ATPase-like responsive cell as determined in vivo, or in vitro,according to standard assay techniques. An ATPase-like activity can be adirect activity, such as an association with or an enzymatic activity ona second protein, or an indirect activity, such as a cellular signalingactivity mediated by interaction of the ATPase-like protein with asecond protein. In a preferred embodiment, an ATPase-like activityincludes at least one or more of the following activities: (1)modulating (stimulating and/or enhancing or inhibiting) cellulardivision; (2) modulating organelle biogenesis; (3) modulating proteinsorting; (4) modulating gene expression; (5) modulating proteindegradation; and (6) modulating the function of the 26S proteosome.

An “isolated” or “purified” ATPase-like nucleic acid molecule orprotein, or biologically active portion thereof, is substantially freeof other cellular material, or culture medium when produced byrecombinant techniques, or substantially free of chemical precursors orother chemicals when chemically synthesized. Preferably, an “isolated”nucleic acid is free of sequences (preferably protein encodingsequences) that naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For purposes of theinvention, “isolated” when used to refer to nucleic acid moleculesexcludes isolated chromosomes. For example, in various embodiments, theisolated ATPase-like nucleic acid molecule can contain less than about 5kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequencesthat naturally flank the nucleic acid molecule in genomic DNA of thecell from which the nucleic acid is derived. An ATPase-like protein thatis substantially free of cellular material includes preparations ofATPase-like protein having less than about 30%, 20%, 10%, or 5% (by dryweight) of non-ATPase protein (also referred to herein as a“contaminating protein”). When the ATPase-like protein or biologicallyactive portion thereof is recombinantly produced, preferably, culturemedium represents less than about 30%, 20%, 10%, or 5% of the volume ofthe protein preparation. When ATPase-like protein is produced bychemical synthesis, preferably the protein preparations have less thanabout 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors ornon-ATPase chemicals.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculescomprising nucleotide sequences encoding ATPase-like proteins andpolypeptides or biologically active portions thereof, as well as nucleicacid molecules sufficient for use as hybridization probes to identifyATPase-like-encoding nucleic acids (e.g., ATPase-like mRNA) andfragments for use as PCR primers for the amplification or mutation ofATPase-like nucleic acid molecules. As used herein, the term “nucleicacid molecule” is intended to include DNA molecules (e.g., cDNA orgenomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

Nucleotide sequences encoding the ATPase-like proteins of the presentinvention include sequences set forth in SEQ ID NO:1, 3, and complementsthereof. By “complement” is intended a nucleotide sequence that issufficiently complementary to a given nucleotide sequence such that itcan hybridize to the given nucleotide sequence to thereby form a stableduplex. The corresponding amino acid sequence for the ATPase-likeprotein encoded by these nucleotide sequences is set forth in SEQ IDNO:2. The invention also encompasses nucleic acid molecules comprisingnucleotide sequences encoding partial-length ATPase-like proteins,including the sequence set forth in SEQ ID NO:1, 3, and complementsthereof.

Nucleic acid molecules that are fragments of these ATPase-likenucleotide sequences are also encompassed by the present invention. By“fragment” is intended a portion of the nucleotide sequence encoding anATPase-like protein. A fragment of an ATPase-like nucleotide sequencemay encode a biologically active portion of an ATPase-like protein, orit may be a fragment that can be used as a hybridization probe or PCRprimer using methods disclosed below. A biologically active portion ofan ATPase-like protein can be prepared by isolating a portion of one ofthe ATPase-like nucleotide sequences of the invention, expressing theencoded portion of the ATPase-like protein (e.g., by recombinantexpression in vitro), and assessing the activity of the encoded portionof the ATPase-like protein. Nucleic acid molecules that are fragments ofan ATPase-like nucleotide sequence comprise at least 15, 20, 50, 75,100, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400nucleotides, or up to the number of nucleotides present in a full-lengthATPase-like nucleotide sequence disclosed herein (for example, 1748nucleotides for SEQ ID NO:1 and 1086 nucleotides for SEQ ID NO:3)depending upon the intended use. Alternatively, a nucleic acid moleculesthat is a fragment of an ATPase-like nucleotide sequence of the presentinvention comprises a nucleotide sequence consisting of nucleotides1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800,800-900, 900-1086, 1086-1100, 1100-1200, 1200-1300, 1300-1400,1400-1500, 1500-1600, 1600-1700, or 1700-1748 of SEQ ID NO:1 or 3.

It is understood that isolated fragments include any contiguous sequencenot disclosed prior to the invention as well as sequences that aresubstantially the same and which are not disclosed. Accordingly, if anisolated fragment is disclosed prior to the present invention, thatfragment is not intended to be encompassed by the invention. When asequence is not disclosed prior to the present invention, an isolatednucleic acid fragment is at least about 12, 15, 20, 25, or 30 contiguousnucleotides. Other regions of the nucleotide sequence may comprisefragments of various sizes, depending upon potential homology withpreviously disclosed sequences.

A fragment of an ATPase-like nucleotide sequence that encodes abiologically active portion of an ATPase-like protein of the inventionwill encode at least 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250,or 300 contiguous amino acids, or up to the total number of amino acidspresent in a full-length ATPase-like protein of the invention (forexample, 362 amino acids). Fragments of an ATPase-like nucleotidesequence that are useful as hybridization probes for PCR primersgenerally need not encode a biologically active portion of anATPase-like protein.

Nucleic acid molecules that are variants of the ATPase-like nucleotidesequences disclosed herein are also encompassed by the presentinvention. “Variants” of the ATPase-like nucleotide sequences includethose sequences that encode the ATPase-like proteins disclosed hereinbut that differ conservatively because of the degeneracy of the geneticcode. These naturally occurring allelic variants can be identified withthe use of well-known molecular biology techniques, such as polymerasechain reaction (PCR) and hybridization techniques as outlined below.Variant nucleotide sequences also include synthetically derivednucleotide sequences that have been generated, for example, by usingsite-directed mutagenesis but which still encode the ATPase-likeproteins disclosed in the present invention as discussed below.Generally, nucleotide sequence variants of the invention will have atleast 45%, 55%, 65%, 75%, 85%, 95%, or 98% identity to a particularnucleotide sequence disclosed herein. A variant ATPase-like nucleotidesequence will encode an ATPase-like protein that has an amino acidsequence having at least 45%, 55%, 65%, 75%, 85%, 95%, or 98% identityto the amino acid sequence of an ATPase-like protein disclosed herein.

In addition to the ATPase-like nucleotide sequences shown in SEQ IDNOS:1 and 3, it will be appreciated by those skilled in the art that DNAsequence polymorphisms that lead to changes in the amino acid sequencesof ATPase-like proteins may exist within a population (e.g., the humanpopulation). Such genetic polymorphism in an ATPase-like gene may existamong individuals within a population due to natural allelic variation.An allele is one of a group of genes that occur alternatively at a givengenetic locus. As used herein, the terms “gene” and “recombinant gene”refer to nucleic acid molecules comprising an open reading frameencoding an ATPase-like protein, preferably a mammalian ATPase-likeprotein. As used herein, the phrase “allelic variant” refers to anucleotide sequence that occurs at an ATPase-like locus or to apolypeptide encoded by the nucleotide sequence. Such natural allelicvariations can typically result in 1-5% variance in the nucleotidesequence of the ATPase-like gene. Any and all such nucleotide variationsand resulting amino acid polymorphisms or variations in an ATPase-likesequence that are the result of natural allelic variation and that donot alter the functional activity of ATPase-like proteins are intendedto be within the scope of the invention.

Moreover, nucleic acid molecules encoding ATPase-like proteins fromother species (ATPase-like homologues), which have a nucleotide sequencediffering from that of the ATPase-like sequences disclosed herein, areintended to be within the scope of the invention. For example, nucleicacid molecules corresponding to natural allelic variants and homologuesof the human ATPase-like cDNA of the invention can be isolated based ontheir identity to the human ATPase-like nucleic acid disclosed hereinusing the human cDNA, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under stringenthybridization conditions as disclosed below.

In addition to naturally-occurring allelic variants of the ATPase-likesequences that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequences of the invention thereby leading to changes in theamino acid sequence of the encoded ATPase-like proteins, withoutaltering the biological activity of the ATPase-like proteins. Thus, anisolated nucleic acid molecule encoding an ATPase-like protein having asequence that differs from that of SEQ ID NO:2 can be created byintroducing one or more nucleotide substitutions, additions, ordeletions into the corresponding nucleotide sequence disclosed herein,such that one or more amino acid substitutions, additions or deletionsare introduced into the encoded protein. Mutations can be introduced bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Such variant nucleotide sequences are also encompassed bythe present invention.

For example, preferably, conservative amino acid substitutions may bemade at one or more predicted, preferably nonessential amino acidresidues. A “nonessential” amino acid residue is a residue that can bealtered from the wild-type sequence of an ATPase-like protein (e.g., thesequence of SEQ ID NO:2) without altering the biological activity,whereas an “essential” amino acid residue is required for biologicalactivity. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Such substitutions would not be made for conserved aminoacid residues, or for amino acid residues residing within a conservedmotif, such as the growth factor and cytokine receptor signature 2sequence and the U-PAR/Ly-6 domain sequence of SEQ ID NO:2, where suchresidues are essential for protein activity.

Alternatively, variant ATPase-like nucleotide sequences can be made byintroducing mutations randomly along all or part of an ATPase-likecoding sequence, such as by saturation mutagenesis, and the resultantmutants can be screened for ATPase-like biological activity to identifymutants that retain activity. Following mutagenesis, the encoded proteincan be expressed recombinantly, and the activity of the protein can bedetermined using standard assay techniques.

Thus the nucleotide sequences of the invention include the sequencesdisclosed herein as well as fragments and variants thereof. TheATPase-like nucleotide sequences of the invention, and fragments andvariants thereof, can be used as probes and/or primers to identifyand/or clone ATPase-like homologues in other cell types, e.g., fromother tissues, as well as ATPase-like homologues from other mammals.Such probes can be used to detect transcripts or genomic sequencesencoding the same or identical proteins. These probes can be used aspart of a diagnostic test kit for identifying cells or tissues thatmisexpress an ATPase-like protein, such as by measuring levels of anATPase-like-encoding nucleic acid in a sample of cells from a subject,e.g., detecting ATPase-like mRNA levels or determining whether a genomicATPase-like gene has been mutated or deleted.

In this manner, methods such as PCR, hybridization, and the like can beused to identify such sequences having substantial identity to thesequences of the invention. See, for example, Sambrook et al. (1989)Molecular Cloning: Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.) and Innis, et al. (1990) PCRProtocols: A Guide to Methods and Applications (Academic Press, NY).ATPase-like nucleotide sequences isolated based on their sequenceidentity to the ATPase-like nucleotide sequences set forth herein or tofragments and variants thereof are encompassed by the present invention.

In a hybridization method, all or part of a known ATPase-like nucleotidesequence can be used to screen cDNA or genomic libraries. Methods forconstruction of such cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.). The so-called hybridization probes may be genomic DNAfragments, cDNA fragments, RNA fragments, or other oligonucleotides, andmay be labeled with a detectable group such as ³²P, or any otherdetectable marker, such as other radioisotopes, a fluorescent compound,an enzyme, or an enzyme co-factor. Probes for hybridization can be madeby labeling synthetic oligonucleotides based on the known ATPase-likenucleotide sequence disclosed herein. Degenerate primers designed on thebasis of conserved nucleotides or amino acid residues in a knownATPase-like nucleotide sequence or encoded amino acid sequence canadditionally be used. The probe typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 50, 75, 100,125, 150, 175, 200, 250, 300, 350, or 400 consecutive nucleotides of anATPase-like nucleotide sequence of the invention or a fragment orvariant thereof. Preparation of probes for hybridization is generallyknown in the art and is disclosed in Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.), herein incorporated by reference.

For example, in one embodiment, a previously unidentified ATPase-likenucleic acid molecule hybridizes under stringent conditions to a probethat is a nucleic acid molecule comprising one of the ATPase-likenucleotide sequences of the invention or a fragment thereof. In anotherembodiment, the previously unknown ATPase-like nucleic acid molecule isat least 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700,800, 900, 1000, 2,000, 3,000, 4,000 or 5,000 nucleotides in length andhybridizes under stringent conditions to a probe that is a nucleic acidmolecule comprising one of the ATPase-like nucleotide sequencesdisclosed herein or a fragment thereof.

Accordingly, in another embodiment, an isolated previously unknownATPase-like nucleic acid molecule of the invention is at least 300, 325,350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1,100,1,200, 1,300, or 1,400 nucleotides in length and hybridizes understringent conditions to a probe that is a nucleic acid moleculecomprising one of the nucleotide sequences of the invention, preferablythe coding sequence set forth in SEQ ID NO:1, 3, or a complement,fragment, or variant thereof.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences typically remain hybridized to each other.Such stringent conditions are known to those skilled in the art and canbe found in Current Protocols in Molecular Biology (John Wiley & Sons,New York (1989)), 6.3.1-6.3.6. A preferred, example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50° C. Another example of stringent hybridizationconditions are hybridization in 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at55° C. A further example of stringent hybridization conditions arehybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.Preferably, stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 65° C. Particularly preferredstringency conditions (and the conditions that should be used if thepractitioner is uncertain about what conditions should be applied todetermine if a molecule is within a hybridization limitation of theinvention) are 0.5M Sodium Phosphate, 7% SDS at 65° C., followed by oneor more washes at 0.2×SSC, 1% SDS at 65° C. Preferably, an isolatednucleic acid molecule that hybridizes under stringent conditions to anATPase-like sequence of the invention corresponds to anaturally-occurring nucleic acid molecule. As used herein, a“naturally-occurring” nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural protein).

Thus, in addition to the ATPase-like nucleotide sequences disclosedherein and fragments and variants thereof, the isolated nucleic acidmolecules of the invention also encompass homologous DNA sequencesidentified and isolated from other cells and/or organisms byhybridization with entire or partial sequences obtained from theATPase-like nucleotide sequences disclosed herein or variants andfragments thereof.

The present invention also encompasses antisense nucleic acid molecules,i.e., molecules that are complementary to a sense nucleic acid encodinga protein, e.g., complementary to the coding strand of a double-strandedcDNA molecule, or complementary to an mRNA sequence. Accordingly, anantisense nucleic acid can hydrogen bond to a sense nucleic acid. Theantisense nucleic acid can be complementary to an entire ATPase-likecoding strand, or to only a portion thereof, e.g., all or part of theprotein coding region (or open reading frame). An antisense nucleic acidmolecule can be antisense to a noncoding region of the coding strand ofa nucleotide sequence encoding an ATPase-like protein. The noncodingregions are the 5′ and 3′ sequences that flank the coding region and arenot translated into amino acids.

Given the coding-strand sequence encoding an ATPase-like proteindisclosed herein (e.g., SEQ ID NO:1), antisense nucleic acids of theinvention can be designed according to the rules of Watson and Crickbase pairing. The antisense nucleic acid molecule can be complementaryto the entire coding region of ATPase-like mRNA, but more preferably isan oligonucleotide that is antisense to only a portion of the coding ornoncoding region of ATPase-like mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of ATPase-like mRNA. An antisense oligonucleotidecan be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50nucleotides in length. An antisense nucleic acid of the invention can beconstructed using chemical synthesis and enzymatic ligation proceduresknown in the art.

For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, including, but not limited to, for example e.g., phosphorothioatederivatives and acridine substituted nucleotides. Alternatively, theantisense nucleic acid can be produced biologically using an expressionvector into which a nucleic acid has been subcloned in an antisenseorientation (i.e., RNA transcribed from the inserted nucleic acid willbe of an antisense orientation to a target nucleic acid of interest,described further in the following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding an ATPase-likeprotein to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. An example of a route ofadministration of antisense nucleic acid molecules of the inventionincludes direct injection at a tissue site. Alternatively, antisensenucleic acid molecules can be modified to target selected cells and thenadministered systemically. For example, antisense molecules can belinked to peptides or antibodies to form a complex that specificallybinds to receptors or antigens expressed on a selected cell surface. Theantisense nucleic acid molecules can also be delivered to cells usingthe vectors described herein. To achieve sufficient intracellularconcentrations of the antisense molecules, vector constructs in whichthe antisense nucleic acid molecule is placed under the control of astrong pol II or pol m promoter are preferred.

An antisense nucleic acid molecule of the invention can be an α-anomericnucleic acid molecule. An α-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

The invention also encompasses ribozymes, which are catalytic RNAmolecules with ribonuclease activity that are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Ribozymes (e.g., hammerhead ribozymes (describedin Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used tocatalytically cleave ATPase-like mRNA transcripts to thereby inhibittranslation of ATPase-like mRNA. A ribozyme having specificity for anATPase-like-encoding nucleic acid can be designed based upon thenucleotide sequence of an ATPase-like cDNA disclosed herein (e.g., SEQID NO:1). See, e.g., Cech et al., U.S. Pat. No. 4,987,071; and Cech etal., U.S. Pat. No. 5,116,742. Alternatively, ATPase-like mRNA can beused to select a catalytic RNA having a specific ribonuclease activityfrom a pool of RNA molecules. See, e.g., Bartel and Szostak (1993)Science 261:1411-1418.

The invention also encompasses nucleic acid molecules that form triplehelical structures. For example, ATPase-like gene expression can beinhibited by targeting nucleotide sequences complementary to theregulatory region of the ATPase-like protein (e.g., the ATPase-likepromoter and/or enhancers) to form triple helical structures thatprevent transcription of the ATPase-like gene in target cells. Seegenerally Helene (1991) Anticancer Drug Des. 6(6):569; Helene (1992)Ann. N.Y. Acad. Sci. 660:27; and Maher (1992) Bioassays 14(12):807.

In preferred embodiments, the nucleic acid molecules of the inventioncan be modified at the base moiety, sugar moiety, or phosphate backboneto improve, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal. (1996) Bioorganic & Medicinal Chemistry 4:5). As used herein, theterms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics,e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid-phase peptide synthesis protocols as described in Hyrupet al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci.USA 93:14670.

PNAs of an ATPase-like molecule can be used in therapeutic anddiagnostic applications. For example, PNAs can be used as antisense orantigene agents for sequence-specific modulation of gene expression by,e.g., inducing transcription or translation arrest or inhibitingreplication. PNAs of the invention can also be used, e.g., in theanalysis of single base pair mutations in a gene by, e.g., PNA-directedPCR clamping; as artificial restriction enzymes when used in combinationwith other enzymes, e.g., S1 nucleases (Hyrup (1996), supra; or asprobes or primers for DNA sequence and hybridization (Hyrup (1996),supra; Perry-O'Keefe et al. (1996), supra).

In another embodiment, PNAs of an ATPase-like molecule can be modified,e.g., to enhance their stability, specificity, or cellular uptake, byattaching lipophilic or other helper groups to PNA, by the formation ofPNA-DNA chimeras, or by the use of liposomes or other techniques of drugdelivery known in the art. The synthesis of PNA-DNA chimeras can beperformed as described in Hyrup (1996), supra; Finn et al. (1996)Nucleic Acids Res. 24(17):3357-63; Mag et al. (1989) Nucleic Acids Res.17:5973; and Peterson et al. (1975) Bioorganic Med. Chem. Lett. 5:1119.

II. Isolated ATPase-like Proteins and Anti-ATPase-Like Antibodies

ATPase-like proteins are also encompassed within the present invention.By “ATPase-like protein” is intended a protein having the amino acidsequence set forth in SEQ ID NO: 2, as well as fragments, biologicallyactive portions, and variants thereof.

“Fragments” or “biologically active portions” include polypeptidefragments suitable for use as immunogens to raise anti-ATPase-likeantibodies. Fragments include peptides comprising amino acid sequencessufficiently identical to or derived from the amino acid sequence of anATPase-like protein, or partial-length protein, of the invention andexhibiting at least one activity of an ATPase-like protein, but whichinclude fewer amino acids than the full-length (SEQ ID NO:2) ATPase-likeprotein disclosed herein. Typically, biologically active portionscomprise a domain or motif with at least one activity of the ATPase-likeprotein. A biologically active portion of an ATPase-like protein can bea polypeptide which is, for example, 10, 25, 50, 100 or more amino acidsin length. Such biologically active portions can be prepared byrecombinant techniques and evaluated for one or more of the functionalactivities of a native ATPase-like protein. As used here, a fragmentcomprises at least 5 contiguous amino acids of SEQ ID NO:2. Theinvention encompasses other fragments, however, such as any fragment inthe protein greater than 6, 7, 8, or 9 amino acids.

By “variants” is intended proteins or polypeptides having an amino acidsequence that is at least about 45%, 55%, 65%, preferably about 75%,85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:2.Variants also include polypeptides encoded by a nucleic acid moleculethat hybridizes to the nucleic acid molecule of SEQ ID NO:2, or acomplement thereof, under stringent conditions. In another embodiment, avariant of an isolated polypeptide of the present invention differs, byat least 1, but less than 5, 10, 20, 50, or 100 amino acid residues fromthe sequence shown in SEQ ID NO:2. If alignment is needed for thiscomparison the sequences should be aligned for maximum identity.“Looped” out sequences from deletions or insertions, or mismatches, areconsidered differences. Such variants generally retain the functionalactivity of the ATPase-like proteins of the invention. Variants includepolypeptides that differ in amino acid sequence due to natural allelicvariation or mutagenesis.

The invention also provides ATPase-like chimeric or fusion proteins. Asused herein, an ATPase-like “chimeric protein” or “fusion protein”comprises an ATPase-like polypeptide operably linked to a non-ATPasepolypeptide. An “ATPase-like polypeptide” refers to a polypeptide havingan amino acid sequence corresponding to an ATPase-like protein, whereasa “non-ATPase-like polypeptide” refers to a polypeptide having an aminoacid sequence corresponding to a protein that is not substantiallyidentical to the ATPase-like protein, e.g., a protein that is differentfrom the ATPase-like protein and which is derived from the same or adifferent organism. Within an ATPase-like fusion protein, theATPase-like polypeptide can correspond to all or a portion of anATPase-like protein, preferably at least one biologically active portionof an ATPase-like protein. Within the fusion protein, the term “operablylinked” is intended to indicate that the ATPase-like polypeptide and thenon-ATPase-like polypeptide are fused in-frame to each other. Thenon-ATPase-like polypeptide can be fused to the N-terminus or C-terminusof the ATPase-like polypeptide.

One useful fusion protein is a GST-ATPase-like fusion protein in whichthe ATPase-like sequences are fused to the C-terminus of the GSTsequences. Such fusion proteins can facilitate the purification ofrecombinant ATPase-like proteins.

In yet another embodiment, the fusion protein is anATPase-like-immunoglobulin fusion protein in which all or part of anATPase-like protein is fused to sequences derived from a member of theimmunoglobulin protein family. The ATPase-like-immunoglobulin fusionproteins of the invention can be incorporated into pharmaceuticalcompositions and administered to a subject to inhibit an interactionbetween an ATPase-like ligand and an ATPase-like protein on the surfaceof a cell, thereby suppressing ATPase-like-mediated signal transductionin vivo. The ATPase-like-immunoglobulin fusion proteins can be used toaffect the bioavailability of an ATPase-like cognate ligand. Inhibitionof the ATPase-like ligand/ATPase-like interaction may be usefultherapeutically, both for treating proliferative and differentiativedisorders and for modulating (e.g., promoting or inhibiting) cellsurvival. Moreover, the ATPase-like-immunoglobulin fusion proteins ofthe invention can be used as immunogens to produce anti-ATPase-likeantibodies in a subject, to purify ATPase-like ligands, and in screeningassays to identify molecules that inhibit the interaction of anATPase-like protein with an ATPase-like ligand.

Preferably, an ATPase-like chimeric or fusion protein of the inventionis produced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences may be ligatedtogether in-frame, or the fusion gene can be synthesized, such as withautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers that give rise tocomplementary overhangs between two consecutive gene fragments, whichcan subsequently be annealed and reamplified to generate a chimeric genesequence (see, e.g., Ausubel et al., eds. (1995) Current Protocols inMolecular Biology) (Greene Publishing and Wiley-Interscience, NY).Moreover, an ATPase-like-encoding nucleic acid can be cloned into acommercially available expression vector such that it is linked in-frameto an existing fusion moiety. Variants of the ATPase-like proteins canfunction as either ATPase-like agonists (mimetics) or as ATPase-likeantagonists. Variants of the ATPase-like protein can be generated bymutagenesis, e.g., discrete point mutation or truncation of theATPase-like protein. An agonist of the ATPase-like protein can retainsubstantially the same, or a subset, of the biological activities of thenaturally occurring form of the ATPase-like protein. An antagonist ofthe ATPase-like protein can inhibit one or more of the activities of thenaturally occurring form of the ATPase-like protein by, for example,competitively binding to a downstream or upstream member of a cellularsignaling cascade that includes the ATPase-like protein. Thus, specificbiological effects can be elicited by treatment with a variant oflimited function. Treatment of a subject with a variant having a subsetof the biological activities of the naturally occurring form of theprotein can have fewer side effects in a subject relative to treatmentwith the naturally occurring form of the ATPase-like proteins.

Variants of an ATPase-like protein that function as either ATPase-likeagonists or as ATPase-like antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of anATPase-like protein for ATPase-like protein agonist or antagonistactivity. In one embodiment, a variegated library of ATPase-likevariants is generated by combinatorial mutagenesis at the nucleic acidlevel and is encoded by a variegated gene library. A variegated libraryof ATPase-like variants can be produced by, for example, enzymaticallyligating a mixture of synthetic oligonucleotides into gene sequencessuch that a degenerate set of potential ATPase-like sequences isexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g., for phage display) containing the set ofATPase-like sequences therein. There are a variety of methods that canbe used to produce libraries of potential ATPase-like variants from adegenerate oligonucleotide sequence. Chemical synthesis of a degenerategene sequence can be performed in an automatic DNA synthesizer, and thesynthetic gene then ligated into an appropriate expression vector. Useof a degenerate set of genes allows for the provision, in one mixture,of all of the sequences encoding the desired set of potentialATPase-like sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidRes. 11:477).

In addition, libraries of fragments of an ATPase-like protein codingsequence can be used to generate a variegated population of ATPase-likefragments for screening and subsequent selection of variants of anATPase-like protein. In one embodiment, a library of coding sequencefragments can be generated by treating a double-stranded PCR fragment ofan ATPase-like coding sequence with a nuclease under conditions whereinnicking occurs only about once per molecule, denaturing thedouble-stranded DNA, renaturing the DNA to form double-stranded DNAwhich can include sense/antisense pairs from different nicked products,removing single-stranded portions from reformed duplexes by treatmentwith S1 nuclease, and ligating the resulting fragment library into anexpression vector. By this method, one can derive an expression librarythat encodes N-terminal and internal fragments of various sizes of theATPase-like protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of ATPase-like proteins. Themost widely used techniques, which are amenable to high through-putanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquethat enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify ATPase-likevariants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).

An isolated ATPase-like polypeptide of the invention can be used as animmunogen to generate antibodies that bind ATPase-like proteins usingstandard techniques for polyclonal and monoclonal antibody preparation.The full-length ATPase-like protein can be used or, alternatively, theinvention provides antigenic peptide fragments of ATPase-like proteinsfor use as immunogens. The antigenic peptide of an ATPase-like proteincomprises at least 8, preferably 10, 15, 20, or 30 amino acid residuesof the amino acid sequence shown in SEQ ID NO:2 and encompasses anepitope of an ATPase-like protein such that an antibody raised againstthe peptide forms a specific immune complex with the ATPase-likeprotein. Preferred epitopes encompassed by the antigenic peptide areregions of a ATPase-like protein that are located on the surface of theprotein, e.g., hydrophilic regions.

Accordingly, another aspect of the invention pertains toanti-ATPase-like polyclonal and monoclonal antibodies that bind anATPase-like protein. Polyclonal anti-ATPase-like antibodies can beprepared by immunizing a suitable subject (e.g., rabbit, goat, mouse, orother mammal) with an ATPase-like immunogen. The anti-ATPase-likeantibody titer in the immunized subject can be monitored over time bystandard techniques, such as with an enzyme linked immunosorbent assay(ELISA) using immobilized ATPase-like protein. At an appropriate timeafter immunization, e.g., when the anti-ATPase-like antibody titers arehighest, antibody-producing cells can be obtained from the subject andused to prepare monoclonal antibodies by standard techniques, such asthe hybridoma technique originally described by Kohler and Milstein(1975) Nature 256:495-497, the human B cell hybridoma technique (Kozboret al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole etal. (1985) in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld andSell (Alan R. Liss, Inc., New York, N.Y.), pp. 77-96) or triomatechniques. The technology for producing hybridomas is well known (seegenerally Coligan et al., eds. (1994) Current Protocols in Immunology(John Wiley & Sons, Inc., New York, N.Y.); Galfre et al. (1977) Nature266:55052; Kenneth (1980) in Monoclonal Antibodies: A New Dimension InBiological Analyses (Plenum Publishing Corp., NY; and Lerner (1981) YaleJ. Biol. Med., 54:387-402).

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-ATPase-like antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with an ATPase-like protein to therebyisolate immunoglobulin library members that bind the ATPase-likeprotein. Kits for generating and screening phage display libraries arecommercially available (e.g., the Pharmacia Recombinant Phage AntibodySystem, Catalog No. 27-9400-01; and the Stratagene SurfZAP™ PhageDisplay Kit, Catalog No. 240612). Additionally, examples of methods andreagents particularly amenable for use in generating and screeningantibody display library can be found in, for example, U.S. Pat. No.5,223,409; PCT Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791;WO 92/15679; 93/01288; WO 92/01047; 92/09690; and 90/02809; Fuchs et al.(1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod.Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffithset al. (1993) EMBO J. 12:725-734.

Additionally, recombinant anti-ATPase-like antibodies, such as chimericand humanized monoclonal antibodies, comprising both human and nonhumanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in PCT PublicationNos. WO 86101533 and WO 87/02671; European Patent Application Nos.184,187, 171, 496, 125,023, and 173,494; U.S. Pat. Nos. 4,816,567 and5,225,539; European Patent Application 125,023; Better et al. (1988)Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987)Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw etal. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison (1985) Science229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice that are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. See, for example, Lonberg and Huszar (1995)Int. Rev. Immunol. 13:65-93); and U.S. Pat. Nos. 5,625,126; 5,633,425;5,569,825; 5,661,016; and 5,545,806. In addition, companies such asAbgenix, Inc. (Freemont, Calif.), can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. This technology is described by Jespers etal. (1994) Bio/Technology 12:899-903).

An anti-ATPase-like antibody (e.g., monoclonal antibody) can be used toisolate ATPase-like proteins by standard techniques, such as affinitychromatography or immunoprecipitation. An anti-ATPase-like antibody canfacilitate the purification of natural ATPase-like protein from cellsand of recombinantly produced ATPase-like protein expressed in hostcells. Moreover, an anti-ATPase-like antibody can be used to detectATPase-like protein (e.g., in a cellular lysate or cell supernatant) inorder to evaluate the abundance and pattern of expression of theATPase-like protein. Anti-ATPase-like antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling theantibody to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S,or ³H.

Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). The conjugates of the invention canbe used for modifying a given biological response, the drug moiety isnot to be construed as limited to classical chemical therapeutic agents.For example, the drug moiety may be a protein or polypeptide possessinga desired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, alpha-interferon,beta-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL- 1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies'84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding an ATPase-likeprotein (or a portion thereof). “Vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked, such as a “plasmid”, a circular double-stranded DNA loopinto which additional DNA segments can be ligated, or a viral vector,where additional DNA segments can be ligated into the viral genome. Thevectors are useful for autonomous replication in a host cell or may beintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome (e.g.,nonepisomal mammalian vectors). Expression vectors are capable ofdirecting the expression of genes to which they are operably linked. Ingeneral, expression vectors of utility in recombinant DNA techniques areoften in the form of plasmids (vectors).

However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses, and adeno-associated viruses), that serveequivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell. This means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, operably linked to the nucleicacid sequence to be expressed. “Operably linked” is intended to meanthat the nucleotide sequence of interest is linked to the regulatorysequence(s) in a manner that allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in ahost cell when the vector is introduced into the host cell). The term“regulatory sequence” is intended to include promoters, enhancers, andother expression control elements (e.g., polyadenylation signals). See,for example, Goeddel (1990) in Gene Expression Technology: Methods inEnzymology 185 (Academic Press, San Diego, Calif.). Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cell and those that direct expression ofthe nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., ATPase-like proteins, mutantforms of ATPase-like proteins, fusion proteins, etc.). It is furtherrecognized that the nucleic acid sequences of the invention can bealtered to contain codons, which are preferred, or non preferred, for aparticular expression system. For example, the nucleic acid can be onein which at least one altered codon, and preferably at least 10%, or 20%of the codons have been altered such that the sequence is optimized forexpression in E. coli, yeast, human, insect, or CHO cells. Methods fordetermining such codon usage are well known in the art.

The recombinant expression vectors of the invention can be designed forexpression of ATPase-like protein in prokaryotic or eukaryotic hostcells. Expression of proteins in prokaryotes is most often carried outin E. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or nonfusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Typical fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly,Mass.), and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein. Examples of suitableinducible nonfusion E. coli expression vectors include pTrc (Amann etal. (1988) Gene 69:301-315) and pET 11d (Studier et al. (1990) in GeneExpression Technology: Methods in Enzymology 185 (Academic Press, SanDiego, Calif.), pp. 60-89). Strategies to maximize recombinant proteinexpression in E. coli can be found in Gottesman (1990) in GeneExpression Technology: Methods in Enzymology 185 (Academic Press, CA),pp. 119-128 and Wada et al. (1992) Nucleic Acids Res. 20:2111-2118.Target gene expression from the pTrc vector relies on host RNApolymerase transcription from a hybrid trp-lac fusion promoter.

Suitable eukaryotic host cells include insect cells (examples ofBaculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39)); yeast cells (examples of vectorsfor expression in yeast S. cereivisiae include pYepSec1 (Baldari et al.(1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2(Invitrogen Corporation, San Diego, Calif.), and pPicZ (InvitrogenCorporation, San Diego, Calif.)); or mammalian cells (mammalianexpression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC(Kaufman et al. (1987) EMBO J. 6:187:195)). Suitable mammalian cellsinclude Chinese hamster ovary cells (CHO) or COS cells. In mammaliancells, the expression vector's control functions are often provided byviral regulatory elements. For example, commonly used promoters arederived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus40. For other suitable expression systems for both prokaryotic andeukaryotic cells, see chapters 16 and 17 of Sambrook et al. (1989)Molecular cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.). See, Goeddel (1990) in GeneExpression Technology: Methods in Enzymology 185 (Academic Press, SanDiego, Calif.). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell but are stillincluded within the scope of the term as used herein. A “purifiedpreparation of cells”, as used herein, refers to, in the case of plantor animal cells, an in vitro preparation of cells and not an entireintact plant or animal. In the case of cultured cells or microbialcells, it consists of a preparation of at least 10% and more preferably50% of the subject cells.

In one embodiment, the expression vector is a recombinant mammalianexpression vector that comprises tissue-specific regulatory elementsthat direct expression of the nucleic acid preferentially in aparticular cell type. Suitable tissue-specific promoters include thealbumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev.1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv.Immunol. 43:235-275), in particular promoters of T cell receptors(Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins(Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell33:741-748), neuron-specific promoters (e.g., the neurofilamentpromoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science230:912-916), and mammary gland-specific promoters (e.g., milk wheypromoter; U.S. Pat. No. 4,873,316 and European Application PatentPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379), the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546), and the like.

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperably linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to ATPase-like mRNA. Regulatory sequences operablylinked to a nucleic acid cloned in the antisense orientation can bechosen to direct the continuous expression of the antisense RNA moleculein a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen to direct constitutive,tissue-specific, or cell-type-specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid, or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub et al. (1986)Reviews—Trends in Genetics, Vol. 1(1).

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.) and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin, and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding an ATPase-like protein or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) ATPase-likeprotein. Accordingly, the invention further provides methods forproducing ATPase-like protein using the host cells of the invention. Inone embodiment, the method comprises culturing the host cell of theinvention, into which a recombinant expression vector encoding anATPase-like protein has been introduced, in a suitable medium such thatATPase-like protein is produced. In another embodiment, the methodfurther comprises isolating ATPase-like protein from the medium or thehost cell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichATPase-like-coding sequences have been introduced. Such host cells canthen be used to create nonhuman transgenic animals in which exogenousATPase-like sequences have been introduced into their genome orhomologous recombinant animals in which endogenous ATPase-like sequenceshave been altered. Such animals are useful for studying the functionand/or activity of ATPase-like genes and proteins and for identifyingand/or evaluating modulators of ATPase-like activity. As used herein, a“transgenic animal” is a nonhuman animal, preferably a mammal, morepreferably a rodent such as a rat or mouse, in which one or more of thecells of the animal includes a transgene. Other examples of transgenicanimals include nonhuman primates, sheep, dogs, cows, goats, chickens,amphibians, etc. A transgene is exogenous DNA that is integrated intothe genome of a cell from which a transgenic animal develops and whichremains in the genome of the mature animal, thereby directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic animal. As used herein, a “homologousrecombinant animal” is a nonhuman animal, preferably a mammal, morepreferably a mouse, in which an endogenous ATPase-like gene has beenaltered by homologous recombination between the endogenous gene and anexogenous DNA molecule introduced into a cell of the animal, e.g., anembryonic cell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducingATPase-like-encoding nucleic acid into the male pronuclei of afertilized oocyte, e.g., by microinjection, retroviral infection, andallowing the oocyte to develop in a pseudopregnant female foster animal.The ATPase-like cDNA sequence can be introduced as a transgene into thegenome of a nonhuman animal. Alternatively, a homologue of the mouseATPase-like gene can be isolated based on hybridization and used as atransgene. Intronic sequences and polyadenylation signals can also beincluded in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operablylinked to the ATPase-like transgene to direct expression of ATPase-likeprotein to particular cells. Methods for generating transgenic animalsvia embryo manipulation and microinjection, particularly animals such asmice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866, 4,870,009, and 4,873,191 and inHogan (1986) Manipulating the Mouse Embryo (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods areused for production of other transgenic animals. A transgenic founderanimal can be identified based upon the presence of the ATPase-liketransgene in its genome and/or expression of ATPase-like mRNA in tissuesor cells of the animals. A transgenic founder animal can then be used tobreed additional animals carrying the transgene. Moreover, transgenicanimals carrying a transgene encoding ATPase-like gene can further bebred to other transgenic animals carrying other transgenes.

To create a homologous recombinant animal, one prepares a vectorcontaining at least a portion of an ATPase-like gene or a homolog of thegene into which a deletion, addition, or substitution has beenintroduced to thereby alter, e.g., functionally disrupt, the ATPase-likegene. In a preferred embodiment, the vector is designed such that, uponhomologous recombination, the endogenous ATPase-like gene isfunctionally disrupted (i.e., no longer encodes a functional protein;also referred to as a “knock out” vector). Alternatively, the vector canbe designed such that, upon homologous recombination, the endogenousATPase-like gene is mutated or otherwise altered but still encodesfunctional protein (e.g., the upstream regulatory region can be alteredto thereby alter the expression of the endogenous ATPase-like protein).In the homologous recombination vector, the altered portion of theATPase-like gene is flanked at its 5′ and 3′ ends by additional nucleicacid of the ATPase-like gene to allow for homologous recombination tooccur between the exogenous ATPase-like gene carried by the vector andan endogenous ATPase-like gene in an embryonic stem cell. The additionalflanking ATPase-like nucleic acid is of sufficient length for successfulhomologous recombination with the endogenous gene. Typically, severalkilobases of flanking DNA (both at the 5′ and 3′ ends) are included inthe vector (see, e.g., Thomas and Capecchi (1987) Cell 51:503 for adescription of homologous recombination vectors). The vector isintroduced into an embryonic stem cell line (e.g., by electroporation),and cells in which the introduced ATPase-like gene has homologouslyrecombined with the endogenous ATPase-like gene are selected (see, e.g.,Li et al. (1992) Cell 69:915). The selected cells are then injected intoa blastocyst of an animal (e.g., a mouse) to form aggregation chimeras(see, e.g., Bradley (1987) in Teratocarcinomas and Embryonic Stem Cells:A Practical Approach, ed. Robertson (IRL, Oxford pp. 113-152). Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term. Progeny harboringthe homologously recombined DNA in their germ cells can be used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA by germline transmission of the transgene. Methods forconstructing homologous recombination vectors and homologous recombinantanimals are described further in Bradley (1991) Current Opinion inBio/Technology 2:823-829 and in PCT Publication Nos. WO 90/11354, WO91/01140, WO 92/0968, and WO 93/04169.

In another embodiment, transgenic nonhuman animals containing selectedsystems that allow for regulated expression of the transgene can beproduced. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355). If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the nonhuman transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and PCT Publication Nos. WO 97/07668 and WO 97/07669.

IV. Pharmaceutical Compositions

The ATPase-like nucleic acid molecules, ATPase-like proteins, andanti-ATPase-like antibodies (also referred to herein as “activecompounds”) of the invention can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise the nucleic acid molecule, protein, or antibody and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The compositions of the invention are useful to treat any of thedisorders discussed herein. The compositions are provided intherapeutically effective amounts. By “therapeutically effectiveamounts” is intended an amount sufficient to modulate the desiredresponse. As defined herein, a therapeutically effective amount ofprotein or polypeptide (i.e., an effective dosage) ranges from about0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg bodyweight, more preferably about 0.1 to 20 mg/kg body weight, and even morepreferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7mg/kg, or 5 to 6 mg/kg body weight.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of a protein, polypeptide, or antibody can include asingle treatment or, preferably, can include a series of treatments. Ina preferred example, a subject is treated with antibody, protein, orpolypeptide in the range of between about 0.1 to 20 mg/kg body weight,one time per week for between about 1 to 10 weeks, preferably between 2to 8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody, protein, or polypeptide used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays as described herein.

The present invention encompasses agents which modulate expression oractivity. An agent may, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e,. including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds.

It is understood that appropriate doses of small molecule agents dependsupon a number of factors within the knowledge of the ordinarily skilledphysician, veterinarian, or researcher. The dose(s) of the smallmolecule will vary, for example, depending upon the identity, size, andcondition of the subject or sample being treated, further depending uponthe route by which the composition is to be administered, if applicable,and the effect which the practitioner desires the small molecule to haveupon the nucleic acid or polypeptide of the invention. Exemplary dosesinclude milligram or microgram amounts of the small molecule perkilogram of subject or sample weight (e.g., about 1 microgram perkilogram to about 500 milligrams per kilogram, about 100 micrograms perkilogram to about 5 milligrams per kilogram, or about 1 microgram perkilogram to about 50 micrograms per kilogram. It is furthermoreunderstood that appropriate doses of a small molecule depend upon thepotency of the small molecule with respect to the expression or activityto be modulated. Such appropriate doses may be determined using theassays described herein. When one or more of these small molecules is tobe administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes, or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF; Parsippany, N.J.), or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion, and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., an ATPase-like protein or anti-ATPase-like antibody) inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying, which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth, or gelatin; an excipientsuch as starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. For administrationby inhalation, the compounds are delivered in the form of an aerosolspray from a pressurized container or dispenser that contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art. Thecompounds can also be prepared in the form of suppositories (e.g., withconventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated with each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. Depending on thetype and severity of the disease, about 1 μg/kg to about 15 mg/kg (e.g.,0.1 to 20 mg/kg) of antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to about 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays. Anexemplary dosing regimen is disclosed in WO 94/04188. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470), or by stereotactic injection(see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods:(a) screening assays; (b) detection assays (e.g., chromosomal mapping,tissue typing, forensic biology); (c) predictive medicine (e.g.,diagnostic assays, prognostic assays, monitoring clinical trials, andpharmacogenomics); and (d) methods of treatment (e.g., therapeutic andprophylactic). The isolated nucleic acid molecules of the invention canbe used to express ATPase-like protein (e.g., via a recombinantexpression vector in a host cell in gene therapy applications), todetect ATPase-like mRNA (e.g., in a biological sample) or a geneticlesion in an ATPase-like gene, and to modulate ATPase-like activity. Inaddition, the ATPase-like proteins can be used to screen drugs orcompounds that modulate the cellular activities described above as wellas to treat disorders characterized by insufficient or excessiveproduction of ATPase-like protein or production of ATPase-like proteinforms that have decreased or aberrant activity compared to ATPase-likewild type protein. In addition, the anti-ATPase-like antibodies of theinvention can be used to detect and isolate ATPase-like proteins andmodulate ATPase-like activity.

A. Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules, or otherdrugs) that bind to ATPase-like proteins or have a stimulatory orinhibitory effect on, for example, ATPase-like expression or ATPase-likeactivity.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including biological libraries, spatially addressable parallelsolid phase or solution phase libraries, synthetic library methodsrequiring deconvolution, the “one-bead one-compound” library method, andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, nonpeptide oligomer, orsmall molecule libraries of compounds (Lam (1997) Anticancer Drug Des.12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andGallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Bio/Techniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Pat.No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA89:1865-1869), or phage (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. USA 87:6378-6382; and Felici (1991) J. Mol. Biol.222:301-310).

Determining the ability of the test compound to bind to the ATPase-likeprotein can be accomplished, for example, by coupling the test compoundwith a radioisotope or enzymatic label such that binding of the testcompound to the ATPase-like protein or biologically active portionthereof can be determined by detecting the labeled compound in acomplex. For example, test compounds can be labeled with 125I, ³⁵S, ¹⁴C,or ³H, either directly or indirectly, and the radioisotope detected bydirect counting of radioemmission or by scintillation counting.Alternatively, test compounds can be enzymatically labeled with, forexample, horseradish peroxidase, alkaline phosphatase, or luciferase,and the enzymatic label detected by determination of conversion of anappropriate substrate to product.

In a similar manner, one may determine the ability of the ATPase-likeprotein to bind to or interact with an ATPase-like target molecule. By“target molecule” is intended a molecule with which an ATPase-likeprotein binds or interacts in nature. In a preferred embodiment, theability of the ATPase-like protein to bind to or interact with anATPase-like target molecule can be determined by monitoring the activityof the target molecule. For example, the activity of the target moleculecan be monitored by detecting alterations in protein sorting, celldivision, protein degradation, organelle biogenesis, etc., detectingcatalytic/enzymatic activity of the target on an appropriate substrate,detecting the induction of a reporter gene (e.g., anATPase-like-responsive regulatory element operably linked to a nucleicacid encoding a detectable marker, e.g., luciferase), or detecting acellular response, for example, cellular differentiation or cellproliferation.

In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting an ATPase-like protein orbiologically active portion thereof with a test compound and determiningthe ability of the test compound to bind to the ATPase-like protein orbiologically active portion thereof. Binding of the test compound to theATPase-like protein can be determined either directly or indirectly asdescribed above. In a preferred embodiment, the assay includescontacting the ATPase-like protein or biologically active portionthereof with a known compound that binds ATPase-like protein to form anassay mixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to preferentially bind toATPase-like protein or biologically active portion thereof as comparedto the known compound.

In another embodiment, an assay is a cell-free assay comprisingcontacting ATPase-like protein or biologically active portion thereofwith a test compound and determining the ability of the test compound tomodulate (e.g., stimulate or inhibit) the activity of the ATPase-likeprotein or biologically active portion thereof. Determining the abilityof the test compound to modulate the activity of an ATPase-like proteincan be accomplished, for example, by determining the ability of theATPase-like protein to bind to an ATPase-like target molecule asdescribed above for determining direct binding. In an alternativeembodiment, determining the ability of the test compound to modulate theactivity of an ATPase-like protein can be accomplished by determiningthe ability of the ATPase-like protein to further modulate anATPase-like target molecule. For example, the catalytic/enzymaticactivity of the target molecule on an appropriate substrate can bedetermined as previously described.

In yet another embodiment, the cell-free assay comprises contacting theATPase-like protein or biologically active portion thereof with a knowncompound that binds an ATPase-like protein to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to preferentially bind to or modulate theactivity of an ATPase-like target molecule.

In the above-mentioned assays, it may be desirable to immobilize eitheran ATPase-like protein or its target molecule to facilitate separationof complexed from uncomplexed forms of one or both of the proteins, aswell as to accommodate automation of the assay. In one embodiment, afusion protein can be provided that adds a domain that allows one orboth of the proteins to be bound to a matrix. For example,glutathione-S-transferase/ATPase-like fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione-derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the nonadsorbed targetprotein or ATPase-like protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components andcomplex formation is measured either directly or indirectly, forexample, as described above. Alternatively, the complexes can bedissociated from the matrix, and the level of ATPase-like binding oractivity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, eitherATPase-like protein or its target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated ATPase-likemolecules or target molecules can be prepared frombiotin-NHS(N-hydroxy-succinimide) using techniques well known in the art(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96-well plates (PierceChemicals). Alternatively, antibodies reactive with an ATPase-likeprotein or target molecules but which do not interfere with binding ofthe ATPase-like protein to its target molecule can be derivatized to thewells of the plate, and unbound target or ATPase-like protein trapped inthe wells by antibody conjugation. Methods for detecting such complexes,in addition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with theATPase-like protein or target molecule, as well as enzyme-linked assaysthat rely on detecting an enzymatic activity associated with theATPase-like protein or target molecule.

In another embodiment, modulators of ATPase-like expression areidentified in a method in which a cell is contacted with a candidatecompound and the expression of ATPase-like mRNA or protein in the cellis determined relative to expression of ATPase-like mRNA or protein in acell in the absence of the candidate compound. When expression isgreater (statistically significantly greater) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of ATPase-like mRNA or protein expression.Alternatively, when expression is less (statistically significantlyless) in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of ATPase-like mRNA orprotein expression. The level of ATPase-like mRNA or protein expressionin the cells can be determined by methods described herein for detectingATPase-like mRNA or protein.

In yet another aspect of the invention, the ATPase-like proteins can beused as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Bio/Techniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and PCT Publication No. WO 94/10300), to identify otherproteins, which bind to or interact with ATPase-like protein(“ATPase-like-binding proteins” or “ATPase-like-bp”) and modulateATPase-like activity. Such ATPase-like-binding proteins are also likelyto be involved in the propagation of signals by the ATPase-like proteinsas, for example, upstream or downstream elements of the ATPase-likepathway.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(1) map their respective genes on a chromosome; (2) identify anindividual from a minute biological sample (tissue typing); and (3) aidin forensic identification of a biological sample. These applicationsare described in the subsections below.

1. Chromosome Mapping

The isolated complete or partial ATPase-like gene sequences of theinvention can be used to map their respective ATPase-like genes on achromosome, thereby facilitating the location of gene regions associatedwith genetic disease. Computer analysis of ATPase-like sequences can beused to rapidly select PCR primers (preferably 15-25 bp in length) thatdo not span more than one exon in the genomic DNA, thereby simplifyingthe amplification process. These primers can then be used for PCRscreening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the ATPase-like sequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow (because they lack a particular enzyme), but in whichhuman cells can, the one human chromosome that contains the geneencoding the needed enzyme will be retained. By using various media,panels of hybrid cell lines can be established. Each cell line in apanel contains either a single human chromosome or a small number ofhuman chromosomes, and a full set of mouse chromosomes, allowing easymapping of individual genes to specific human chromosomes (D'Eustachioet al. (1983) Science 220:919-924). Somatic cell hybrids containing onlyfragments of human chromosomes can also be produced by using humanchromosomes with translocations and deletions.

Other mapping strategies that can similarly be used to map anATPase-like sequence to its chromosome include in situ hybridization(described in Fan et al. (1990) Proc. Natl. Acad. Sci. USA 87:6223-27),pre-screening with labeled flow-sorted chromosomes, and pre-selection byhybridization to chromosome specific cDNA libraries. Furthermore,fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. For a review of this technique, seeVerma eta a. (1988) Human Chromosomes: A Manual of Basic Techniques(Pergamon Press, NY). The FISH technique can be used with a DNA sequenceas short as 500 or 600 bases. However, clones larger than 1,000 baseshave a higher likelihood of binding to a unique chromosomal locationwith sufficient signal intensity for simple detection. Preferably 1,000bases, and more preferably 2,000 bases will suffice to get good resultsin a reasonable amount of time.

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Another strategy to map the chromosomal location of ATPase-like genesuses ATPase-like polypeptides and fragments and sequences of the presentinvention and antibodies specific thereto. This mapping can be carriedout by specifically detecting the presence of a ATPase-like polypeptidein members of a panel of somatic cell hybrids between cells of a firstspecies of animal from which the protein originates and cells from asecond species of animal, and then determining which somatic cellhybrid(s) expresses the polypeptide and noting the chromosomes(s) fromthe first species of animal that it contains. For examples of thistechnique, see Pajunen et al. (1988) Cytogenet. Cell. Genet. 47:37-41and Van Keuren et al. (1986) Hum. Genet. 74:34-40. Alternatively, thepresence of a ATPase-like polypeptide in the somatic cell hybrids can bedetermined by assaying an activity or property of the polypeptide, forexample, enzymatic activity, as described in Bordelon-Riser et al.(1979) Somatic Cell Genetics 5:597-613 and Owerbach et al. (1978) Proc.Natl. Acad. Sci. USA 75:5640-5644.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship betweengenes and disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, e.g., Egeland et al. (1987) Nature325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the ATPase-like gene canbe determined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

2. Tissue Typing

The ATPase-like sequences of the present invention can also be used toidentify individuals from minute biological samples. The United Statesmilitary, for example, is considering the use of restriction fragmentlength polymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes and probed on a Southern blot to yield unique bandsfor identification. The sequences of the present invention are useful asadditional DNA markers for RFLP (described in U.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique for determining the actual base-by-baseDNA sequence of selected portions of an individual's genome. Thus, theATPase-like sequences of the invention can be used to prepare two PCRprimers from the 5′ and 3′ ends of the sequences. These primers can thenbe used to amplify an individual's DNA and subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The ATPase-like sequences of the invention uniquelyrepresent portions of the human genome. Allelic variation occurs to somedegree in the coding regions of these sequences, and to a greater degreein the noncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency of about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. The noncoding sequences of SEQ ID NO:1 cancomfortably provide positive individual identification with a panel ofperhaps 10 to 1,000 primers that each yield a noncoding amplifiedsequence of 100 bases. If a predicted coding sequence, such as that inSEQ ID NO:2, is used, a more appropriate number of primers for positiveindividual identification would be 500 to 2,000.

3. Use of Partial ATPase-Like Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. In this manner, PCR technology can be used to amplify DNAsequences taken from very small biological samples such as tissues,e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen foundat a crime scene. The amplified sequence can then be compared to astandard, thereby allowing identification of the origin of thebiological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” that is unique to a particular individual. Asmentioned above, actual base sequence information can be used foridentification as an accurate alternative to patterns formed byrestriction enzyme generated fragments. Sequences targeted to noncodingregions of SEQ ID NO:1 are particularly appropriate for this use asgreater numbers of polymorphisms occur in the noncoding regions, makingit easier to differentiate individuals using this technique. Examples ofpolynucleotide reagents include the ATPase-like sequences or portionsthereof, e.g., fragments derived from the noncoding regions of SEQ IDNO:1 having a length of at least 20 or 30 bases.

The ATPase-like sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes thatcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue. This can be very useful in cases where aforensic pathologist is presented with a tissue of unknown origin.Panels of such ATPase-like probes, can be used to identify tissue byspecies and/or by organ type.

In a similar fashion, these reagents, e.g., ATPase-like primers orprobes can be used to screen tissue culture for contamination (i.e.,screen for the presence of a mixture of different types of cells in aculture).

C. Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trails are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. These applications aredescribed in the subsections below.

1. Diagnostic Assays

One aspect of the present invention relates to diagnostic assays fordetecting ATPase-like protein and/or nucleic acid expression as well asATPase-like activity, in the context of a biological sample. Anexemplary method for detecting the presence or absence of ATPase-likeproteins in a biological sample involves obtaining a biological samplefrom a test subject and contacting the biological sample with a compoundor an agent capable of detecting ATPase-like protein or nucleic acid(e.g., mRNA, genomic DNA) that encodes ATPase-like protein such that thepresence of ATPase-like protein is detected in the biological sample.Results obtained with a biological sample from the test subject may becompared to results obtained with a biological sample from a controlsubject.

“Misexpression or aberrant expression”, as used herein, refers to anon-wild type pattern of gene expression, at the RNA or protein level.It includes: expression at non-wild type levels, i.e., over or underexpression; a pattern of expression that differs from wild type in termsof the time or stage at which the gene is expressed, e.g., increased ordecreased expression (as compared with wild type) at a predetermineddevelopmental period or stage; a pattern of expression that differs fromwild type in terms of decreased expression (as compared with wild type)in a predetermined cell type or tissue type; a pattern of expressionthat differs from wild type in terms of the splicing size, amino acidsequence, post-transitional modification, or biological activity of theexpressed polypeptide; a pattern of expression that differs from wildtype in terms of the effect of an environmental stimulus orextracellular stimulus on expression of the gene, e.g., a pattern ofincreased or decreased expression (as compared with wild type) in thepresence of an increase or decrease in the strength of the stimulus.

A preferred agent for detecting ATPase-like mRNA or genomic DNA is alabeled nucleic acid probe capable of hybridizing to ATPase-like mRNA orgenomic DNA. The nucleic acid probe can be, for example, a full-lengthATPase-like nucleic acid, such as the nucleic acid of SEQ ID NOS:1, 3,or a portion thereof, such as a nucleic acid molecule of at least 15,30, 50, 100, 250, or 500 nucleotides in length and sufficient tospecifically hybridize under stringent conditions to ATPase-like mRNA orgenomic DNA. Other suitable probes for use in the diagnostic assays ofthe invention are described herein.

A preferred agent for detecting ATPase-like protein is an antibodycapable of binding to ATPase-like protein, preferably an antibody with adetectable label. Antibodies can be polyclonal, or more preferably,monoclonal. An intact antibody, or a fragment thereof (e.g., Fab orF(abN)₂) can be used. The term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected with fluorescentlylabeled streptavidin.

The term “biological sample” is intended to include tissues, cells, andbiological fluids isolated from a subject, as well as tissues, cells,and fluids present within a subject. That is, the detection method ofthe invention can be used to detect ATPase-like mRNA, protein, orgenomic DNA in a biological sample in vitro as well as in vivo. Forexample, in vitro techniques for detection of ATPase-like mRNA includeNorthern hybridizations and in situ hybridizations. In vitro techniquesfor detection of ATPase-like protein include enzyme linked immunosorbentassays (ELISAs), Western blots, immunoprecipitations, andimmunofluorescence. In vitro techniques for detection of ATPase-likegenomic DNA include Southern hybridizations. Furthermore, in vivotechniques for detection of ATPase-like protein include introducing intoa subject a labeled anti-ATPase-like antibody. For example, the antibodycan be labeled with a radioactive marker whose presence and location ina subject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject.

The invention also encompasses kits for detecting the presence ofATPase-like proteins in a biological sample (a test sample). Such kitscan be used to determine if a subject is suffering from or is atincreased risk of developing a disorder associated with aberrantexpression of ATPase-like protein. For example, the kit can comprise alabeled compound or agent capable of detecting ATPase-like protein ormRNA in a biological sample and means for determining the amount of anATPase-like protein in the sample (e.g., an anti-ATPase-like antibody oran oligonucleotide probe that binds to DNA encoding an ATPase-likeprotein, e.g., SEQ ID NOS:1 and 3). Kits can also include instructionsfor observing that the tested subject is suffering from or is at risk ofdeveloping a disorder associated with aberrant expression of ATPase-likesequences if the amount of ATPase-like protein or mRNA is above or belowa normal level.

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) that binds to ATPase-likeprotein; and, optionally, (2) a second, different antibody that binds toATPase-like protein or the first antibody and is conjugated to adetectable agent. For oligonucleotide-based kits, the kit can comprise,for example: (1) an oligonucleotide, e.g., a detectably labeledoligonucleotide, that hybridizes to an ATPase-like nucleic acid sequenceor (2) a pair of primers useful for amplifying an ATPase-like nucleicacid molecule.

The kit can also comprise, e.g., a buffering agent, a preservative, or aprotein stabilizing agent. The kit can also comprise componentsnecessary for detecting the detectable agent (e.g., an enzyme or asubstrate). The kit can also contain a control sample or a series ofcontrol samples that can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container, and all of the various containers are within asingle package along with instructions for observing whether the testedsubject is suffering from or is at risk of developing a disorderassociated with aberrant expression of ATPase-like proteins.

2. Other Diagnostic Assays

In another aspect, the invention features, a method of analyzing aplurality of capture probes. The method can be used, e.g., to analyzegene expression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g., anucleic acid or peptide sequence; contacting the array with anATPase-like, preferably purified, nucleic acid, preferably purified,polypeptide, preferably purified, or antibody, and thereby evaluatingthe plurality of capture probes. Binding, e.g., in the case of a nucleicacid, hybridization with a capture probe at an address of the plurality,is detected, e.g., by signal generated from a label attached to theATPase-like nucleic acid, polypeptide, or antibody.

The capture probes can be a set of nucleic acids from a selected sample,e.g., a sample of nucleic acids derived from a control or non-stimulatedtissue or cell.

The method can include contacting the ATPase-like nucleic acid,polypeptide, or antibody with a first array having a plurality ofcapture probes and a second array having a different plurality ofcapture probes. The results of each hybridization can be compared, e.g.,to analyze differences in expression between a first and second sample.The first plurality of capture probes can be from a control sample,e.g., a wild type, normal, or non-diseased, non-stimulated, sample,e.g., a biological fluid, tissue, or cell sample. The second pluralityof capture probes can be from an experimental sample, e.g., a mutanttype, at risk, disease-state or disorder-state, or stimulated, sample,e.g., a biological fluid, tissue, or cell sample.

The plurality of capture probes can be a plurality of nucleic acidprobes each of which specifically hybridizes, with an allele ofATPase-like. Such methods can be used to diagnose a subject, e.g., toevaluate risk for a disease or disorder, to evaluate suitability of aselected treatment for a subject, to evaluate whether a subject has adisease or disorder.

The method can be used to detect SNPs, as described above.

In another aspect, the invention features, a method of analyzing aplurality of probes. The method is useful, e.g., for analyzing geneexpression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the pluralityhaving a unique capture probe, e.g., wherein the capture probes are froma cell or subject which express ATPase-like or from a cell or subject inwhich an ATPase-like mediated response has been elicited, e.g., bycontact of the cell with ATPase-like nucleic acid or protein, oradministration to the cell or subject ATPase-like nucleic acid orprotein; contacting the array with one or more inquiry probe, wherein aninquiry probe can be a nucleic acid, polypeptide, or antibody (which ispreferably other than ATPase-like nucleic acid, polypeptide, orantibody); providing a two dimensional array having a plurality ofaddresses, each address of the plurality being positionallydistinguishable from each other address of the plurality, and eachaddress of the plurality having a unique capture probe, e.g., whereinthe capture probes are from a cell or subject which does not expressATPase-like (or does not express as highly as in the case of theATPase-like positive plurality of capture probes) or from a cell orsubject which in which an ATPase-like mediated response has not beenelicited (or has been elicited to a lesser extent than in the firstsample); contacting the array with one or more inquiry probes (which ispreferably other than an ATPase-like nucleic acid, polypeptide, orantibody), and thereby evaluating the plurality of capture probes.Binding, e.g., in the case of a nucleic acid, hybridization with acapture probe at an address of the plurality, is detected, e.g., bysignal generated from a label attached to the nucleic acid, polypeptide,or antibody.

In another aspect, the invention features, a method of analyzingATPase-like, e.g., analyzing structure, function, or relatedness toother nucleic acid or amino acid sequences. The method includes:providing an ATPase-like nucleic acid or amino acid sequence, e.g., anucleotide sequence from 300-1916 or a portion thereof; comparing theATPase-like sequence with one or more preferably a plurality ofsequences from a collection of sequences, e.g., a nucleic acid orprotein sequence database; to thereby analyze ATPase-like.

The method can include evaluating the sequence identity between anATPase-like sequence and a database sequence. The method can beperformed by accessing the database at a second site, e.g., over theinternet.

In another aspect, the invention features, a set of oligonucleotides,useful, e.g., for identifying SNP's, or identifying specific alleles ofan ATPase-like gene. The set includes a plurality of oligonucleotides,each of which has a different nucleotide at an interrogation position,e.g., an SNP or the site of a mutation. In a preferred embodiment, theoligonucleotides of the plurality identical in sequence with one another(except for differences in length). The oligonucleotides can be providedwith differential labels, such that an oligonucleotides which hybridizesto one allele provides a signal that is distinguishable from anoligonucleotides which hybridizes to a second allele.

3. Prognostic Assays

The methods described herein can furthermore be utilized as diagnosticor prognostic assays to identify subjects having or at risk ofdeveloping a disease or disorder associated with ATPase-like protein,ATPase-like nucleic acid expression, or ATPase-like activity. Prognosticassays can be used for prognostic or predictive purposes to therebyprophylactically treat an individual prior to the onset of a disordercharacterized by or associated with ATPase-like protein, ATPase-likenucleic acid expression, or ATPase-like activity.

Thus, the present invention provides a method in which a test sample isobtained from a subject, and ATPase-like protein or nucleic acid (e.g.,mRNA, genomic DNA) is detected, wherein the presence of ATPase-likeprotein or nucleic acid is diagnostic for a subject having or at risk ofdeveloping a disease or disorder associated with aberrant ATPase-likeexpression or activity. As used herein, a “test sample” refers to abiological sample obtained from a subject of interest. For example, atest sample can be a biological fluid (e.g., serum), cell sample, ortissue.

Furthermore, using the prognostic assays described herein, the presentinvention provides methods for determining whether a subject can beadministered a specific agent (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, nucleic acid, small molecule, or otherdrug candidate) or class of agents (e.g., agents of a type that decreaseATPase-like activity) to effectively treat a disease or disorderassociated with aberrant ATPase-like expression or activity. In thismanner, a test sample is obtained and ATPase-like protein or nucleicacid is detected. The presence of ATPase-like protein or nucleic acid isdiagnostic for a subject that can be administered the agent to treat adisorder associated with aberrant ATPase-like expression or activity.

The methods of the invention can also be used to detect genetic lesionsor mutations in an ATPase-like gene, thereby determining if a subjectwith the lesioned gene is at risk for a disorder characterized byaberrant cellular processes including, altered protein sorting, alteredgene expression, altered cell division, altered protein stability, etc.In preferred embodiments, the methods include detecting, in a sample ofcells from the subject, the presence or absence of a genetic lesion ormutation characterized by at least one of an alteration affecting theintegrity of a gene encoding an ATPase-like-protein, or themisexpression of the ATPase-like gene. For example, such genetic lesionsor mutations can be detected by ascertaining the existence of at leastone of: (1) a deletion of one or more nucleotides from an ATPase-likegene; (2) an addition of one or more nucleotides to an ATPase-like gene;(3) a substitution of one or more nucleotides of an ATPase-like gene;(4) a chromosomal rearrangement of an ATPase-like gene; (5) analteration in the level of a messenger RNA transcript of an ATPase-likegene; (6) an aberrant modification of an ATPase-like gene, such as ofthe methylation pattern of the genomic DNA; (7) the presence of anon-wild-type splicing pattern of a messenger RNA transcript of anATPase gene; (8) a non-wild-type level of an ATPase-like-protein; (9) anallelic loss of an ATPase-like gene; and (10) an inappropriatepost-translational modification of an ATPase-like-protein. As describedherein, there are a large number of assay techniques known in the artthat can be used for detecting lesions in an ATPase-like gene. Any celltype or tissue in which ATPase-like proteins are expressed may beutilized in the prognostic assays described herein.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in theATPase-like-gene (see, e.g., Abravaya et al. (1995) Nucleic Acids Res.23:675-682). It is anticipated that PCR and/or LCR may be desirable touse as a preliminary amplification step in conjunction with any of thetechniques used for detecting mutations described herein.

Alternative amplification methods include self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in an ATPase-like gene from asample cell can be identified by alterations in restriction enzymecleavage patterns of isolated test sample and control DNA digested withone or more restriction endonucleases. Moreover, the use of sequencespecific ribozymes (see, e.g., U.S. Pat. No. 5,498,531) can be used toscore for the presence of specific mutations by development or loss of aribozyme cleavage site.

In other embodiments, genetic mutations in an ATPase-like molecule canbe identified by hybridizing a sample and control nucleic acids, e.g.,DNA or RNA, to high density arrays containing hundreds or thousands ofoligonucleotides probes (Cronin et al. (1996) Human Mutation 7:244-255;Kozal et al. (1996) Nature Medicine 2:753-759). In yet anotherembodiment, any of a variety of sequencing reactions known in the artcan be used to directly sequence the ATPase-like gene and detectmutations by comparing the sequence of the sample ATPase-like gene withthe corresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxim andGilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977)Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any ofa variety of automated sequencing procedures can be utilized whenperforming the diagnostic assays ((1995) Bio/Techniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT PublicationNo. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; andGriffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

Other methods for detecting mutations in the ATPase-like gene includemethods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al.(1985) Science 230:1242). See, also Cotton et al. (1988) Proc. Natl.Acad. Sci. USA 85:4397; Saleeba et al. (1992) Methods Enzymol.217:286-295. In a preferred embodiment, the control DNA or RNA can belabeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more “DNA mismatch repair” enzymes that recognize mismatched basepairs in double-stranded DNA in defined systems for detecting andmapping point mutations in ATPase-like cDNAs obtained from samples ofcells. See, e.g., Hsu et al. (1994) Carcinogenesis 15:1657-1662.According to an exemplary embodiment, a probe based on an ATPase-likesequence, e.g., a wild-type ATPase-like sequence, is hybridized to acDNA or other DNA product from a test cell(s). The duplex is treatedwith a DNA mismatch repair enzyme, and the cleavage products, if any,can be detected from electrophoresis protocols or the like. See, e.g.,U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in ATPase-like genes. For example,single-strand conformation polymorphism (SSCP) may be used to detectdifferences in electrophoretic mobility between mutant and wild-typenucleic acids (Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766;see also Cotton (1993) Mutat. Res. 285:125-144; Hayashi (1992) Genet.Anal. Tech. Appl. 9:73-79). The sensitivity of the assay may be enhancedby using RNA (rather than DNA), in which the secondary structure is moresensitive to a change in sequence. In a preferred embodiment, thesubject method utilizes heteroduplex analysis to separatedouble-stranded heteroduplex molecules on the basis of changes inelectrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditions thatpermit hybridization only if a perfect match is found (Saiki et al.(1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci. USA86:6230). Such allele-specific oligonucleotides are hybridized toPCR-amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele-specific amplification technology, which dependson selective PCR amplification, may be used in conjunction with theinstant invention. Oligonucleotides used as primers for specificamplification may carry the mutation of interest in the center of themolecule so that amplification depends on differential hybridization(Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme3N end of one primer where, under appropriate conditions, mismatch canprevent or reduce polymerase extension (Prossner (1993) Tibtech 11:238).In addition, it may be desirable to introduce a novel restriction sitein the region of the mutation to create cleavage-based detection(Gasparini et al. (1992) Mol. Cell Probes 6: 1). It is anticipated thatin certain embodiments amplification may also be performed using Taqligase for amplification (Barany (1991) Proc. Natl. Acad. Sci. USA88:189). In such cases, ligation will occur only if there is a perfectmatch at the 3N end of the SN sequence making it possible to detect thepresence of a known mutation at a specific site by looking for thepresence or absence of amplification.

The methods described herein may be performed, for example, by utilizingprepackaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnosed patients exhibiting symptoms orfamily history of a disease or illness involving an ATPase-like gene.

4. Pharmacogenomics

Agents, or modulators that have a stimulatory or inhibitory effect onATPase-like activity (e.g., ATPase-like gene expression) as identifiedby a screening assay described herein, can be administered toindividuals to treat (prophylactically or therapeutically) disordersassociated with aberrant ATPase-like activity as well as to modulate acellular phenotype associated with aberrant ATPase-like activity. Inconjunction with such treatment, the pharmacogenomics (i.e., the studyof the relationship between an individual's genotype and thatindividual's response to a foreign compound or drug) of the individualmay be considered. Differences in metabolism of therapeutics can lead tosevere toxicity or therapeutic failure by altering the relation betweendose and blood concentration of the pharmacologically active drug. Thus,the pharmacogenomics of the individual permits the selection ofeffective agents (e.g., drugs) for prophylactic or therapeutictreatments based on a consideration of the individual's genotype. Suchpharmacogenomics can further be used to determine appropriate dosagesand therapeutic regimens. Accordingly, the activity of ATPase-likeprotein, expression of ATPase-like nucleic acid, or mutation content ofATPase-like genes in an individual can be determined to thereby selectappropriate agent(s) for therapeutic or prophylactic treatment of theindividual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Linder (1997) Clin. Chem.43(2):254-266. In general, two types of pharmacogenetic conditions canbe differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body are referred to as “altered drugaction.” Genetic conditions transmitted as single factors altering theway the body acts on drugs are referred to as “altered drug metabolism”.These pharmacogenetic conditions can occur either as rare defects or aspolymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency(G6PD) is a common inherited enzymopathy in which the main clinicalcomplication is haemolysis after ingestion of oxidant drugs(antimalarials, sulfonamides, analgesics, nitrofurans) and consumptionof fava beans.

Differences in metabolism of therapeutics can lead to severe toxicity ortherapeutic failure by altering the relation between dose and bloodconcentration of the pharmacologically active drug. Thus, a physician orclinician may consider applying knowledge obtained in relevantpharmacogenomics studies in determining whether to administer aATPase-like molecule or ATPase-like modulator as well as tailoring thedosage and/or therapeutic regimen of treatment with a ATPase-likemolecule or ATPase-like modulator.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drug's target is known (e.g., anATPase-like protein of the present invention), all common variants ofthat gene can be fairly easily identified in the population and it canbe determined if having one version of the gene versus another isassociated with a particular drug response.

Alternatively, a method termed the “gene expression profiling”, can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., an ATPase-likemolecule or ATPase-like modulator of the present invention) can give anindication whether gene pathways related to toxicity have been turnedon.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment of an individual.This knowledge, when applied to dosing or drug selection, can avoidadverse reactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with an ATPase-likemolecule or ATPase-like modulator, such as a modulator identified by oneof the exemplary screening assays described herein.

The present invention further provides methods for identifying newagents, or combinations, that are based on identifying agents thatmodulate the activity of one or more of the gene products encoded by oneor more of the ATPase-like genes of the present invention, wherein theseproducts may be associated with resistance of the cells to a therapeuticagent. Specifically, the activity of the proteins encoded by theATPase-like genes of the present invention can be used as a basis foridentifying agents for overcoming agent resistance. By blocking theactivity of one or more of the resistance proteins, target cells willbecome sensitive to treatment with an agent that the unmodified targetcells were resistant to.

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a ATPase-like protein can be applied in clinical trials. Forexample, the effectiveness of an agent determined by a screening assayas described herein to increase ATPase-like gene expression, proteinlevels, or upregulate ATPase-like activity, can be monitored in clinicaltrials of subjects exhibiting decreased ATPase-like gene expression,protein levels, or downregulated ATPase-like activity. Alternatively,the effectiveness of an agent determined by a screening assay todecrease ATPase-like gene expression, protein levels, or downregulateATPase-like activity, can be monitored in clinical trials of subjectsexhibiting increased ATPase-like gene expression, protein levels, orupregulated ATPase-like activity. In such clinical trials, theexpression or activity of a ATPase-like gene, and preferably, othergenes that have been implicated in, for example, aATPase-like-associated disorder can be used as a “read out” or markersof the phenotype of a particular cell.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, a PM will show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the activity of ATPase-like protein, expression of ATPase-likenucleic acid, or mutation content of ATPase-like genes in an individualcan be determined to thereby select appropriate agent(s) for therapeuticor prophylactic treatment of the individual. In addition,pharmacogenetic studies can be used to apply genotyping of polymorphicalleles encoding drug-metabolizing enzymes to the identification of anindividual's drug responsiveness phenotype. This knowledge, when appliedto dosing or drug selection, can avoid adverse reactions or therapeuticfailure and thus enhance therapeutic or prophylactic efficiency whentreating a subject with an ATPase-like modulator; such as a modulatoridentified by one of the exemplary screening assays described herein.

5. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of ATPase-like genes can be applied not only inbasic drug screening but also in clinical trials. For example, theeffectiveness of an agent, as determined by a screening assay asdescribed herein, to increase or decrease ATPase-like gene expression,protein levels, or protein activity, can be monitored in clinical trialsof subjects exhibiting decreased or increased ATPase-like geneexpression, protein levels, or protein activity. In such clinicaltrials, ATPase-like expression or activity and preferably that of othergenes that have been implicated in influencing ATPase-like expression oractivity, can be used as a marker of the immune responsiveness of aparticular cell.

For example, and not by way of limitation, genes that are modulated incells by treatment with an agent (e.g., compound, drug, or smallmolecule) that modulates ATPase-like activity (e.g., as identified in ascreening assay described herein) can be identified. Thus, to study theeffect of agents on cellular disorders resulting from aberrantATPase-like activity, for example, in a clinical trial, cells can beisolated and RNA prepared and analyzed for the levels of expression ofATPase-like genes and other genes implicated in the disorder. The levelsof gene expression (i.e., a gene expression pattern) can be quantifiedby Northern blot analysis or RT-PCR, as described herein, oralternatively by measuring the amount of protein produced, by one of themethods as described herein, or by measuring the levels of activity ofATPase-like genes or other genes. In this way, the gene expressionpattern can serve as a marker, indicative of the physiological responseof the cells to the agent. Accordingly, this response state may bedetermined before, and at various points during, treatment of theindividual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (1) obtaininga preadministration sample from a subject prior to administration of theagent;

-   -   (2) detecting the level of expression of an ATPase-like protein,        mRNA, or genomic DNA in the preadministration sample; (3)        obtaining one or more postadministration samples from the        subject; (4) detecting the level of expression or activity of        the ATPase-like protein, mRNA, or genomic DNA in the        postadministration samples; (5) comparing the level of        expression or activity of the ATPase-like protein, mRNA, or        genomic DNA in the preadministration sample with the ATPase-like        protein, mRNA, or genomic DNA in the postadministration sample        or samples; and (vi) altering the administration of the agent to        the subject accordingly to bring about the desired effect, i.e.,        for example, an increase or a decrease in the expression or        activity of an ATPase-like protein.

C. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant ATPase-like expression oractivity. Additionally, the compositions of the invention find use inthe treatment of disorders described herein. Thus, therapies fordisorders associated with ATPase-like molecules are encompassed herein.“Subject”, as used herein, can refer to a mammal, e.g., a human, or toan experimental or animal or disease model. The subject can also be anon-human animal, e.g., a horse, cow, goat, or other domestic animal.

“Treatment” is herein defined as the application or administration of atherapeutic agent to a patient, or application or administration of atherapeutic agent to an isolated tissue or cell line from a patient, whohas a disease, a symptom of disease or a predisposition toward adisease, with the purpose to cure, heal, alleviate, relieve, alter,remedy, ameliorate, improve or affect the disease, the symptoms ofdisease or the predisposition toward disease. A “therapeutic agent”includes, but is not limited to, small molecules, peptides, antibodies,ribozymes and antisense oligonucleotides.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject a disease or condition associated with an aberrant ATPase-likeexpression or activity by administering to the subject an agent thatmodulates ATPase-like expression or at least one ATPase-like geneactivity. Subjects at risk for a disease that is caused, or contributedto, by aberrant ATPase-like expression or activity can be identified by,for example, any or a combination of diagnostic or prognostic assays asdescribed herein. Administration of a prophylactic agent can occur priorto the manifestation of symptoms characteristic of the ATPase-likeaberrancy, such that a disease or disorder is prevented or,alternatively, delayed in its progression. Depending on the type ofATPase-like aberrancy, for example, an ATPase-like agonist orATPase-like antagonist agent can be used for treating the subject. Theappropriate agent can be determined based on screening assays describedherein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulatingATPase-like expression or activity for therapeutic purposes. Themodulatory method of the invention involves contacting a cell with anagent that modulates one or more of the activities of ATPase-likeprotein activity associated with the cell. An agent that modulatesATPase-like protein activity can be an agent as described herein, suchas a nucleic acid or a protein, a naturally-occurring cognate ligand ofan ATPase-like protein, a peptide, an ATPase-like peptidomimetic, orother small molecule. In one embodiment, the agent stimulates one ormore of the biological activities of ATPase-like protein. Examples ofsuch stimulatory agents include active ATPase-like protein and a nucleicacid molecule encoding an ATPase-like protein that has been introducedinto the cell. In another embodiment, the agent inhibits one or more ofthe biological activities of ATPase-like protein. Examples of suchinhibitory agents include antisense ATPase-like nucleic acid moleculesand anti-ATPase-like antibodies.

These modulatory methods can be performed in vitro (e.g., by culturingthe cell with the agent) or, alternatively, in vivo (e.g, byadministering the agent to a subject). As such, the present inventionprovides methods of treating an individual afflicted with a disease ordisorder characterized by aberrant expression or activity of anATPase-like protein or nucleic acid molecule. In one embodiment, themethod involves administering an agent (e.g., an agent identified by ascreening assay described herein), or a combination of agents, thatmodulates (e.g., upregulates or downregulates) ATPase-like expression oractivity. In another embodiment, the method involves administering anATPase-like protein or nucleic acid molecule as therapy to compensatefor reduced or aberrant ATPase-like expression or activity.

Stimulation of ATPase-like activity is desirable in situations in whichan ATPase-like protein is abnormally downregulated and/or in whichincreased ATPase-like activity is likely to have a beneficial effect.Conversely, inhibition of ATPase-like activity is desirable insituations in which ATPase-like activity is abnormally upregulatedand/or in which decreased ATPase-like activity is likely to have abeneficial effect.

This invention is further illustrated by the following examples, whichshould not be construed as limiting.

EXPERIMENTAL Example 1 Tissue Distribution of ATPase-Like mRNA

Northern blot hybridizations with various RNA samples can be performedunder standard conditions and washed under stringent conditions, i.e.,0.2×SSC at 65° C. A DNA probe corresponding to all or a portion of theATPase-like cDNA (SEQ ID NO:1 or 3) can be used. The DNA wasradioactively labeled with ³²P-dCTP using the Prime-It Kit (Stratagene,La Jolla, Calif.) according to the instructions of the supplier. Filterscontaining mRNA from mouse hematopoietic and endocrine tissues, andcancer cell lines (Clontech, Palo Alto, Calif.) can be probed inExpressHyb hybridization solution (Clontech) and washed at highstringency according to manufacturer's recommendations.

TAQMAN analysis of the 7970 sequence revealed expression in a number oftissues as shown in FIG. 4. High levels of 7970 transcripts are seen inthe fetal heart, normal heart, heart (CHF), brain cortex, brainhypothalamus, glial cells, and epithelial cells. Moderate levels ofexpression are found in kidney, fetal liver, aortic SMC early, aorticSMC late, shear HUVEC, and static HUVEC. Low levels of expression werefound in the aorta, vein, spinal cord, brain, gioblastoma, breast,ovary, tumorous breast, tumorous ovary, pancreas, prostate, tumorousprostate, colon, tumorous colon, liver, liver fibrosis, lung, tumorouslung, lung (COPD), spleen, tonsil, lymph node, thymus, endothelial cells(aortic), skeletal muscle, fibroblasts, skin, adipose, osteoblasts(primary), osteoblasts (undifferentiated), osteoblasts (differentiated),and osteoclasts.

The expression of the 7970 sequence in various tumorous and normaltissues was also determined. FIGS. 5-8 and FIGS. 10-12 show the relativeexpression levels of the 7970 transcript in various tissues including,for example, normal and tumorous colon tissues, normal liver andmetastatic liver tissues, normal brain and tumorous brain tissues,normal breast and tumorous breast tissues, normal ovary and tumorousovary tissues, normal lung and tumorous lung tissues.

FIG. 9 further shows the expression level of the 7970 transcript in anon-small cell lung cancer cell line (H640) in the presence and theabsence of the p16 gene.

Example 2 Recombinant Expression of ATPase-Like in Bacterial Cells

In this example, the ATPase-like sequence is expressed as a recombinantglutathione-S-transferase (GST) fusion polypeptide in E. coli and thefusion polypeptide is isolated and characterized. Specifically, theATPase-like sequence is fused to GST and this fusion polypeptide isexpressed in E. coli, e.g., strain PEB199. Expression of theGST-ATPase-like fusion protein in PEB199 is induced with IPTG. Therecombinant fusion polypeptide is purified from crude bacterial lysatesof the induced PEB 199 strain by affinity chromatography on glutathionebeads. Using polyacrylamide gel electrophoretic analysis of thepolypeptide purified from the bacterial lysates, the molecular weight ofthe resultant fusion polypeptide is determined.

Example 3 Expression of Recombinant ATPase-Like Protein in COS Cells

To express the ATPase-like gene in COS cells, the pcDNA/Amp vector byInvitrogen Corporation (San Diego, Calif.) is used. This vector containsan SV40 origin of replication, an ampicillin resistance gene, an E. colireplication origin, a CMV promoter followed by a polylinker region, andan SV40 intron and polyadenylation site. A DNA fragment encoding theentire ATPase-like protein and an HA tag (Wilson et al. (1984) Cell37:767) or a FLAG tag fused in-frame to its 3′ end of the fragment iscloned into the polylinker region of the vector, thereby placing theexpression of the recombinant protein under the control of the CMVpromoter.

To construct the plasmid, the ATPase-like DNA sequence is amplified byPCR using two primers. The 5′ primer contains the restriction site ofinterest followed by approximately twenty nucleotides of the ATPase-likecoding sequence starting from the initiation codon; the 3′ end sequencecontains complementary sequences to the other restriction site ofinterest, a translation stop codon, the HA tag or FLAG tag and the last20 nucleotides of the ATPase-like coding sequence. The PCR amplifiedfragment and the pcDNA/Amp vector are digested with the appropriaterestriction enzymes and the vector is dephosphorylated using the CIAPenzyme (New England Biolabs, Beverly, Mass.). Preferably the tworestriction sites chosen are different so that the ATPase-like gene isinserted in the correct orientation. The ligation mixture is transformedinto E. coli cells (strains HB101, DH5α, SURE, available from StratageneCloning Systems, La Jolla, Calif., can be used), the transformed cultureis plated on ampicillin media plates, and resistant colonies areselected. Plasmid DNA is isolated from transformants and examined byrestriction analysis for the presence of the correct fragment.

COS cells are subsequently transfected with the ATPase-like-pcDNA/Ampplasmid DNA using the calcium phosphate or calcium chlorideco-precipitation methods, DEAE-dextran-mediated transfection,lipofection, or electroporation. Other suitable methods for transfectinghost cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989. The expression of the ATPase-like polypeptide is detected byradiolabelling (³⁵S-methionine or ³⁵S-cysteine available from NEN,Boston, Mass., can be used) and immunoprecipitation (Harlow, E. andLane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1988) using an HA specific monoclonalantibody. Briefly, the cells are labeled for 8 hours with ³⁵S-methionine(or ³⁵S-cysteine). The culture media are then collected and the cellsare lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1%SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culturemedia are precipitated with an HA specific monoclonal antibody.Precipitated polypeptides are then analyzed by SDS-PAGE.

Alternatively, DNA containing the ATPase-like coding sequence is cloneddirectly into the polylinker of the pcDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of theATPase-like polypeptide is detected by radiolabelling andimmunoprecipitation using a ATPase-like specific monoclonal antibody.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

Chapter 2 32670, Novel Human Phosphatidylserine Synthase-Like Moleculesand Uses Thereof BACKGROUND OF THE INVENTION

The membranes of eukaryotic cells contain not only large amounts ofcholesterol but a variety of phospholipids. For example, the majorphospholipids in the human erythrocyte include phosphatidylethanolamine,phosphatidylcholine, phosphatidylserine, and sphingomyelin. The fourmajor phospholipids in the yeast Saccharomyces cerevisiae arephosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylinositol (PI), and phosphatidylserine (PS) (Poole et al.(1986) J. of Bacteriology 168(2):668-672). PS accounts for 4 to 8% ofthe total membrane phospholipids in S. cerevisiae and is important tooverall lipid metabolism (Atkinson et al. (1980) J. Biol. Chem. 255:6653-6661). PS is the normal precursor to PE and PC. The synthesis ofphospholipids in eukaryotic cells involves both cytoplasmic andmembrane-associated enzymes, a number of which are coordinatelyregulated (Henry et al., (1984) Annu. Rev. Genet. 18:207-231).

Phosphatidylserine (PS) is an essential phospholipid for the growth ofmammalian cells, comprising approximately 10% of the total membrane ofvarious mammalian tissues and cultured cells (Kuge et al. (1986) J.Biol. Chem. 261:5790-5794).

The enzyme responsible for the biosynthesis of PS in S. cerevisiae isCDPdiacylglycerol:L-serine O-phoshphatidyl transferase(Phosphatidylserine synthase; EC 2.7.8.8). PS synthase catalyzes theformation of PS and CMP from CDP-diacylglycerol (CDP-DG) and serine by asequential Bi Bi reaction mechanism (Bae-Lee et al. (1984) J. Biol.Chem. 259: 10857-10862). PS synthase is an integral membrane protein.Detailed biochemical studies have shown that PS synthase activity ispresent in both the mitochondria and endoplasmic reticulum (Kuchler etal. (1986) J. Bacteriol. 165:901-910).

Phosphatidylserine (PS) synthase in Chinese Hamster Ovary cells (CHO)exists in two forms—(PSS) I and II. PSS I and PSS II are encoded by twogenes pssA and pssB, respectively (Kuge et al. (1997) J. Biol. Chem.272: 19133-19139). PSS I is responsible for the conversion ofphosphatidylcholine to phosphatidylserine and PSS II is responsible forthe conversion of phosphatidylethanolamine to phosphatidylserine (Saitoet al. (1998) J. Biol. Chem. 273:17199-17205). PS biosynthesis in CHO-K1cells is inhibited upon the addition of PS to the culture mediumsuggesting that feedback control is involved in the regulation of PSbiosynthesis (Nishijima et al. (1986) J. Biol. Chem. 261:5784-5789).

Various CHO mutants have been identified which exhibit defectivesynthesis in PSS I and PSS II. PSS I and PSS II are similar in sequenceto each other; there is a 38% amino acid identity between the two PSsynthases (Kuge et al. (1997) J. Biol. Chem. 272:19133-19139). Resultsobtained by Kuge et al. indicate that PSS II in CHO-K1 cells isinhibited by exogenous PS and that the activity of over-produced PSS IIin CHO-K1 cells is depressed for maintenance of the normal PSbiosynthetic rate, probably through molecular mechanisms different fromthose for the exogenous PS-mediated inhibition. Also, the work of Kugeet al. demonstrated that the ARG-97 residue of PSS II is critical forboth the exogenous PS-mediated inhibition of PS II and the depression ofoverproduced PSS II activity.

Because phospholipids such as PS are important components of eukaryoticmembranes their proper biosynthesis is critical to cell homeostasis andfunction. Defects in PS synthase may yield membranes with alteredfunctionality which could have implications in a wide range of diseasestates. Accordingly, PS synthases are a major target for drug action anddevelopment. Accordingly, it is valuable to the field of pharmaceuticaldevelopment to identify and characterize novel PS synthases and tissuesand disorders in which PS synthases are differentially expressed. Thepresent invention advances the state of the art by providing novel humanPS synthase molecules and the uses thereof.

SUMMARY OF THE INVENTION

Isolated nucleic acid molecules corresponding to humanphosphatidylserine synthase-like nucleic acid sequences are provided.Additionally, amino acid sequences corresponding to the polynucleotidesare encompassed. In particular, the present invention provides forisolated nucleic acid molecules comprising nucleotide sequences encodingthe amino acid sequences shown in SEQ ID NO:6. Further provided arehuman phosphatidylserine synthase-like polypeptides having an amino acidsequence encoded by a nucleic acid molecule described herein.

The present invention also provides vectors and host cells forrecombinant expression of the nucleic acid molecules described herein,as well as methods of making such vectors and host cells and for usingthem for production of the polypeptides or peptides of the invention byrecombinant techniques.

The human phosphatidylserine synthase-like molecules of the presentinvention are useful for modulating the biosynthetic pathway involvingthe synthesis of the membrane phospholipid phosphatidylserine (PS). Themolecules may be useful for a wide variety of human disorders as hereindescribed.

Accordingly, in one aspect, this invention provides isolated nucleicacid molecules encoding human phosphatidylserine synthase-like proteinsor biologically active portions thereof, as well as nucleic acidfragments suitable as primers or hybridization probes for the detectionof human phosphatidylserine synthase-like encoding nucleic acids.

Another aspect of this invention features isolated or recombinant humanphosphatidylserine synthase-like proteins and polypeptides. Preferredhuman phosphatidylserine synthase-like proteins and polypeptides possessat least one biological activity possessed by naturally occurring humanphosphatidylserine synthase-like proteins.

Variant nucleic acid molecules and polypeptides substantially homologousto the nucleotide and amino acid sequences set forth in the sequencelistings are encompassed by the present invention. Additionally,fragments and substantially homologous fragments of the nucleotide andamino acid sequences are provided.

Antibodies and antibody fragments that selectively bind the humanphosphatidylserine synthase-like polypeptides and fragments areprovided. Such antibodies are useful in detecting the humanphosphatidylserine synthase-like polypeptides.

In another aspect, the present invention provides a method for detectingthe presence of human phosphatidylserine synthase-like activity orexpression in a biological sample by contacting the biological samplewith an agent capable of detecting an indicator of humanphosphatidylserine synthase-like activity such that the presence ofhuman phosphatidylserine synthase-like activity is detected in thebiological sample.

In yet another aspect, the invention provides a method for modulatinghuman phosphatidylserine synthase-like activity comprising contacting acell with an agent that modulates (inhibits or stimulates) humanphosphatidylserine synthase-like activity or expression such that humanphosphatidylserine synthase-like activity or expression in the cell ismodulated. In one embodiment, the agent is an antibody that specificallybinds to human phosphatidylserine synthase-like protein. In anotherembodiment, the agent modulates expression of human phosphatidylserinesynthase-like protein by modulating transcription of a humanphosphatidylserine synthase-like gene, splicing of a humanphosphatidylserine synthase-like mRNA, or translation of a humanphosphatidylserine synthase-like mRNA. In yet another embodiment, theagent is a nucleic acid molecule having a nucleotide sequence that isantisense to the coding strand of the human phosphatidylserinesynthase-like mRNA or the human phosphatidylserine synthase-like gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant humanphosphatidylserine synthase-like protein activity or nucleic acidexpression by administering an agent that is a human phosphatidylserinesynthase-like modulator to the subject. In one embodiment, the humanphosphatidylserine synthase-like modulator is a human phosphatidylserinesynthase-like protein. In another embodiment, the humanphosphatidylserine synthase-like modulator is a human phosphatidylserinesynthase-like nucleic acid molecule. In other embodiments, the humanphosphatidylserine synthase-like modulator is a peptide, peptidomimetic,or other small molecule.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic lesion or mutation characterized byat least one of the following: (1) aberrant modification or mutation ofa gene encoding a human phosphatidylserine synthase-like protein; (2)misregulation of a gene encoding a human phosphatidylserinesynthase-like protein; and (3) aberrant post-translational modificationof a human phosphatidylserine synthase-like protein, wherein a wild-typeform of the gene encodes a protein with a human phosphatidylserinesynthase-like activity.

In another aspect, the invention provides a method for identifying acompound that binds to or modulates the activity of a humanphosphatidylserine synthase-like protein. In general, such methodsentail measuring a biological activity of a human phosphatidylserinesynthase-like protein in the presence and absence of a test compound andidentifying those compounds that alter the activity of the humanphosphatidylserine synthase-like protein.

The invention also features methods for identifying a compound thatmodulates the expression of human phosphatidylserine synthase-like genesby measuring the expression of the human phosphatidylserinesynthase-like sequences in the presence and absence of the compound.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the invention are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

The present invention provides phosphatidylserine synthase-likemolecules. By “phosphatidylserine synthase-like molecules” is intended anovel human sequence referred to as 32670, and variants and fragmentsthereof. These full-length gene sequences or fragments thereof arereferred to as “phosphatidylserine synthase-like” sequences indicatingthey share sequence similarity with phosphatidylserine synthase genes.Isolated nucleic acid molecules comprising nucleotide sequences encodingthe 32670 polypeptide whose amino acid sequence is given in SEQ ID NO:6,or a variant or fragment thereof, are provided. A nucleotide sequenceencoding the 32670 polypeptide is set forth in SEQ ID NO:5, and a codingsequence encoding the 32670 polypeptide is set: forth in SEQ ID NO:7.

The disclosed invention relates to methods and compositions for themodulation, diagnosis, and treatment of the disorders of the variouscells and tissues herein described.

Disorders involving the spleen include, but are not limited to,splenomegaly, including nonspecific acute splenitis, congestivespenomegaly, and spenic infarcts; neoplasms, congenital anomalies, andrupture. Disorders associated with splenomegaly include infections, suchas nonspecific splenitis, infectious mononucleosis, tuberculosis,typhoid fever, brucellosis, cytomegalovirus, syphilis, malaria,histoplasmosis, toxoplasmosis, kala-azar, trypanosomiasis,schistosomiasis, leishmaniasis, and echinococcosis; congestive statesrelated to partial hypertension, such as cirrhosis of the liver, portalor splenic vein thrombosis, and cardiac failure; lymphohematogenousdisorders, such as Hodgkin disease, non-Hodgkin lymphomas/leukemia,multiple myeloma, myeloproliferative disorders, hemolytic anemias, andthrombocytopenic purpura; immunologic-inflammatory conditions, such asrheumatoid arthritis and systemic lupus erythematosus; storage diseasessuch as Gaucher disease, Niemann-Pick disease, andmucopolysaccharidoses; and other conditions, such as amyloidosis,primary neoplasms and cysts, and secondary neoplasms.

Disorders involving the lung include, but are not limited to, congenitalanomalies; atelectasis; diseases of vascular origin, such as pulmonarycongestion and edema, including hemodynamic pulmonary edema and edemacaused by microvascular injury, adult respiratory distress syndrome(diffuse alveolar damage), pulmonary embolism, hemorrhage, andinfarction, and pulmonary hypertension and vascular sclerosis; chronicobstructive pulmonary disease, such as emphysema, chronic bronchitis,bronchial asthma, and bronchiectasis; diffuse interstitial(infiltrative, restrictive) diseases, such as pneumoconioses,sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitialpneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia(pulmonary infiltration with eosinophilia), Bronchiolitisobliterans—organizing pneumonia, diffuse pulmonary hemorrhage syndromes,including Goodpasture syndrome, idiopathic pulmonary hemosiderosis andother hemorrhagic syndromes, pulmonary involvement in collagen vasculardisorders, and pulmonary alveolar proteinosis; complications oftherapies, such as drug-induced lung disease, radiation-induced lungdisease, and lung transplantation; tumors, such as bronchogeniccarcinoma, including paraneoplastic syndromes, bronchioloalveolarcarcinoma, neuroendocrine tumors, such as bronchial carcinoid,miscellaneous tumors, and metastatic tumors; pathologies of the pleura,including inflammatory pleural effusions, noninflammatory pleuraleffusions, pneumothorax, and pleural tumors, including solitary fibroustumors (pleural fibroma) and malignant mesothelioma.

Disorders involving the colon include, but are not limited to,congenital anomalies, such as atresia and stenosis, Meckel diverticulum,congenital aganglionic megacolon-Hirschsprung disease; enterocolitis,such as diarrhea and dysentery, infectious enterocolitis, includingviral gastroenteritis, bacterial enterocolitis, necrotizingenterocolitis, antibiotic-associated colitis (pseudomembranous colitis),and collagenous and lymphocytic colitis, miscellaneous intestinalinflammatory disorders, including parasites and protozoa, acquiredimmunodeficiency syndrome, transplantation, drug-induced intestinalinjury, radiation enterocolitis, neutropenic colitis (typhlitis), anddiversion colitis; idiopathic inflammatory bowel disease, such as Crohndisease and ulcerative colitis; tumors of the colon, such asnon-neoplastic polyps, adenomas, familial syndromes, colorectalcarcinogenesis, colorectal carcinoma, and carcinoid tumors.

Disorders involving the liver include, but are not limited to, hepaticinjury; jaundice and cholestasis, such as bilirubin and bile formation;hepatic failure and cirrhosis, such as cirrhosis, portal hypertension,including ascites, portosystemic shunts, and splenomegaly; infectiousdisorders, such as viral hepatitis, including hepatitis A-E infectionand infection by other hepatitis viruses, clinicopathologic syndromes,such as the carrier state, asymptomatic infection, acute viralhepatitis, chronic viral hepatitis, and fulminant hepatitis; autoimmunehepatitis; drug- and toxin-induced liver disease, such as alcoholicliver disease; inborn errors of metabolism and pediatric liver disease,such as hemochromatosis, Wilson disease, α₁-antitrypsin deficiency, andneonatal hepatitis; intrahepatic biliary tract disease, such assecondary biliary cirrhosis, primary biliary cirrhosis, primarysclerosing cholangitis, and anomalies of the biliary tree; circulatorydisorders, such as impaired blood flow into the liver, including hepaticartery compromise and portal vein obstruction and thrombosis, impairedblood flow through the liver, including passive congestion andcentrilobular necrosis and peliosis hepatis, hepatic vein outflowobstruction, including hepatic vein thrombosis (Budd-Chiari syndrome)and veno-occlusive disease; hepatic disease associated with pregnancy,such as preeclampsia and eclampsia, acute fatty liver of pregnancy, andintrehepatic cholestasis of pregnancy; hepatic complications of organ orbone marrow transplantation, such as drug toxicity after bone marrowtransplantation, graft-versus-host disease and liver rejection, andnonimmunologic damage to liver allografts; tumors and tumorousconditions, such as nodular hyperplasias, adenomas, and malignanttumors, including primary carcinoma of the liver and metastatic tumors.

Disorders involving the uterus and endometrium include, but are notlimited to, endometrial histology in the menstrual cycle; functionalendometrial disorders, such as anovulatory cycle, inadequate lutealphase, oral contraceptives and induced endometrial changes, andmenopausal and postmenopausal changes; inflammations, such as chronicendometritis; adenomyosis; endometriosis; endometrial polyps;endometrial hyperplasia; malignant tumors, such as carcinoma of theendometrium; mixed Müllerian and mesenchymal tumors, such as malignantmixed Müllerian tumors; tumors of the myometrium, including leiomyomas,leiomyosarcomas, and endometrial stromal tumors.

Disorders involving the brain include, but are not limited to, disordersinvolving neurons, and disorders involving glia, such as astrocytes,oligodendrocytes, ependymal cells, and microglia; cerebral edema, raisedintracranial pressure and herniation, and hydrocephalus; malformationsand developmental diseases, such as neural tube defects, forebrainanomalies, posterior fossa anomalies, and syringomyelia and hydromyelia;perinatal brain injury; cerebrovascular diseases, such as those relatedto hypoxia, ischemia, and infarction, including hypotension,hypoperfusion, and low-flow states—global cerebral ischemia and focalcerebral ischemia—infarction from obstruction of local blood supply,intracranial hemorrhage, including intracerebral (intraparenchymal)hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, andvascular malformations, hypertensive cerebrovascular disease, includinglacunar infarcts, slit hemorrhages, and hypertensive encephalopathy;infections, such as acute meningitis, including acute pyogenic(bacterial) meningitis and acute aseptic (viral) meningitis, acute focalsuppurative infections, including brain abscess, subdural empyema, andextradural abscess, chronic bacterial meningoencephalitis, includingtuberculosis and mycobacterioses, neurosyphilis, and neuroborreliosis(Lyme disease), viral meningoencephalitis, including arthropod-borne(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes simplexvirus Type 2, Varicalla-zoster virus (Herpes zoster), cytomegalovirus,poliomyelitis, rabies, and human immunodeficiency virus 1, includingHIV-1 meningoencephalitis (subacute encephalitis), vacuolar myelopathy,AIDS-associated myopathy, peripheral neuropathy, and AIDS in children,progressive multifocal leukoencephalopathy, subacute sclerosingpanencephalitis, fungal meningoencephalitis, other infectious diseasesof the nervous system; transmissible spongiform encephalopathies (priondiseases); demyelinating diseases, including multiple sclerosis,multiple sclerosis variants, acute disseminated encephalomyelitis andacute necrotizing hemorrhagic encephalomyelitis, and other diseases withdemyelination; degenerative diseases, such as degenerative diseasesaffecting the cerebral cortex, including Alzheimer disease and Pickdisease, degenerative diseases of basal ganglia and brain stem,including Parkinsonism, idiopathic Parkinson disease (paralysisagitans), progressive supranuclear palsy, corticobasal degeneration,multiple system atrophy, including striatonigral degeneration,Shy-Drager syndrome, and olivopontocerebellar atrophy, and Huntingtondisease; spinocerebellar degenerations, including spinocerebellarataxias, including Friedreich ataxia, and ataxia-telanglectasia,degenerative diseases affecting motor neurons, including amyotrophiclateral sclerosis (motor neuron disease), bulbospinal atrophy (Kennedysyndrome), and spinal muscular atrophy; inborn errors of metabolism,such as leukodystrophies, including Krabbe disease, metachromaticleukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, andCanavan disease, mitochondrial encephalomyopathies, including Leighdisease and other mitochondrial encephalomyopathies; toxic and acquiredmetabolic diseases, including vitamin deficiencies such as thiamine(vitamin B₁) deficiency and vitamin B₁₂ deficiency, neurologic sequelaeof metabolic disturbances, including hypoglycemia, hyperglycemia, andhepatic encephatopathy, toxic disorders, including carbon monoxide,methanol, ethanol, and radiation, including combined methotrexate andradiation-induced injury; tumors, such as gliomas, includingastrocytoma, including fibrillary (diffuse) astrocytoma and glioblastomamultiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, andbrain stem glioma, oligodendroglioma, and ependymoma and relatedparaventricular mass lesions, neuronal tumors, poorly differentiatedneoplasms, including medulloblastoma, other parenchymal tumors,including primary brain lymphoma, germ cell tumors, and pinealparenchymal tumors, meningiomas, metastatic tumors, paraneoplasticsyndromes, peripheral nerve sheath tumors, including schwannoma,neurofibroma, and malignant peripheral nerve sheath tumor (malignantschwannoma), and neurocutaneous syndromes (phakomatoses), includingneurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindaudisease.

Disorders involving T-cells include, but are not limited to,cell-mediated hypersensitivity, such as delayed type hypersensitivityand T-cell-mediated cytotoxicity, and transplant rejection; autoimmunediseases, such as systemic lupus erythematosus, Sjögren syndrome,systemic sclerosis, inflammatory myopathies, mixed connective tissuedisease, and polyarteritis nodosa and other vasculitides; immunologicdeficiency syndromes, including but not limited to, primaryimmunodeficiencies, such as thymic hypoplasia, severe combinedimmunodeficiency diseases, and AIDS; leukopenia; reactive (inflammatory)proliferations of white cells, including but not limited to,leukocytosis, acute nonspecific lymphadenitis, and chronic nonspecificlymphadenitis; neoplastic proliferations of white cells, including butnot limited to lymphoid neoplasms, such as precursor T-cell neoplasms,such as acute lymphoblastic leukemia/lymphoma, peripheral T-cell andnatural killer cell neoplasms that include peripheral T-cell lymphoma,unspecified, adult T-cell leukemia/lymphoma, mycosis fungoides andSezary syndrome, and Hodgkin disease.

Diseases of the skin, include but are not limited to, disorders ofpigmentation and melanocytes, including but not limited to, vitiligo,freckle, melasma, lentigo, nevocellular nevus, dysplastic nevi, andmalignant melanoma; benign epithelial tumors, including but not limitedto, seborrheic keratoses, acanthosis nigricans, fibroepithelial polyp,epithelial cyst, keratoacanthoma, and adnexal (appendage) tumors;premalignant and malignant epidermal tumors, including but not limitedto, actinic keratosis, squamous cell carcinoma, basal cell carcinoma,and merkel cell carcinoma; tumors of the dermis, including but notlimited to, benign fibrous histiocytoma, dermatofibrosarcomaprotuberans, xanthomas, and dermal vascular tumors; tumors of cellularimmigrants to the skin, including but not limited to, histiocytosis X,mycosis fungoides (cutaneous T-cell lymphoma), and mastocytosis;disorders of epidermal maturation, including but not limited to,ichthyosis; acute inflammatory dermatoses, including but not limited to,urticaria, acute eczematous dermatitis, and erythema multiforme; chronicinflammatory dermatoses, including but not limited to, psoriasis, lichenplanus, and lupus erythematosus; blistering (bullous) diseases,including but not limited to, pemphigus, bullous pemphigoid, dermatitisherpetiformis, and noninflammatory blistering diseases: epidermolysisbullosa and porphyria; disorders of epidermal appendages, including butnot limited to, acne vulgaris; panniculitis, including but not limitedto, erythema nodosum and erythema induratum; and infection andinfestation, such as verrucae, molluscum contagiosum, impetigo,superficial fungal infections, and arthropod bites, stings, andinfestations.

In normal bone marrow, the myelocytic series (polymorphoneuclear cells)make up approximately 60% of the cellular elements, and the erythrocyticseries, 20-30%. Lymphocytes, monocytes, reticular cells, plasma cellsand megakaryocytes together constitute 10-20%. Lymphocytes make up 5-15%of normal adult marrow. In the bone marrow, cell types are add mixed sothat precursors of red blood cells (erythroblasts), macrophages(monoblasts), platelets (megakaryocytes), polymorphoneuclear leucocytes(myeloblasts), and lymphocytes (lymphoblasts) can be visible in onemicroscopic field. In addition, stem cells exist for the different celllineages, as well as a precursor stem cell for the committed progenitorcells of the different lineages. The various types of cells and stagesof each would be known to the person of ordinary skill in the art andare found, for example, on page 42 (FIG. 2-8) of Immunology,Imunopathology and Immunity, Fifth Edition, Sell et al. Simon andSchuster (1996), incorporated by reference for its teaching of celltypes found in the bone marrow. According, the invention is directed todisorders arising from these cells. These disorders include but are notlimited to the following: diseases involving hematopoeitic stem cells;committed lymphoid progenitor cells; lymphoid cells including B andT-cells; committed myeloid progenitors, including monocytes,granulocytes, and megakaryocytes; and committed erythroid progenitors.These include but are not limited to the leukemias, including B-lymphoidleukemias, T-lymphoid leukemias, undifferentiated leukemias;erythroleukemia, megakaryoblastic leukemia, monocytic; [leukemias areencompassed with and without differentiation]; chronic and acutelymphoblastic leukemia, chronic and acute lymphocytic leukemia, chronicand acute myelogenous leukemia, lymphoma, myelo dysplastic syndrome,chronic and acute myeloid leukemia, myelomonocytic leukemia; chronic andacute myeloblastic leukemia, chronic and acute myelogenous leukemia,chronic and acute promyelocytic leukemia, chronic and acute myelocyticleukemia, hematologic malignancies of monocyte-macrophage lineage, suchas juvenile chronic myelogenous leukemia; secondary AML, antecedenthematological disorder; refractory anemia; aplastic anemia; reactivecutaneous angioendotheliomatosis; fibrosing disorders involving alteredexpression in dendritic cells, disorders including systemic sclerosis,E-M syndrome, epidemic toxic oil syndrome, eosinophilic fasciitislocalized forms of scleroderma, keloid, and fibrosing colonopathy;angiomatoid malignant fibrous histiocytoma; carcinoma, including primaryhead and neck squamous cell carcinoma; sarcoma, including kaposi'ssarcoma; fibroadanoma and phyllodes tumors, including mammaryfibroadenoma; stromal tumors; phyllodes tumors, including histiocytoma;erythroblastosis; neurofibromatosis; diseases of the vascularendothelium; demyelinating, particularly in old lesions; gliosis,vasogenic edema, vascular disease, Alzheimer's and Parkinson's disease;T-cell lymphomas; B-cell lymphomas.

Disorders involving the heart, include but are not limited to, heartfailure, including but not limited to, cardiac hypertrophy, left-sidedheart failure, and right-sided heart failure; ischemic heart disease,including but not limited to angina pectoris, myocardial infarction,chronic ischemic heart disease, and sudden cardiac death; hypertensiveheart disease, including but not limited to, systemic (left-sided)hypertensive heart disease and pulmonary (right-sided) hypertensiveheart disease; valvular heart disease, including but not limited to,valvular degeneration caused by calcification, such as calcific aorticstenosis, calcification of a congenitally bicuspid aortic valve, andmitral annular calcification, and myxomatous degeneration of the mitralvalve (mitral valve prolapse), rheumatic fever and rheumatic heartdisease, infective endocarditis, and noninfected vegetations, such asnonbacterial thrombotic endocarditis and endocarditis of systemic lupuserythematosus (Libman-Sacks disease), carcinoid heart disease, andcomplications of artificial valves; myocardial disease, including butnot limited to dilated cardiomyopathy, hypertrophic cardiomyopathy,restrictive cardiomyopathy, and myocarditis; pericardial disease,including but not limited to, pericardial effusion and hemopericardiumand pericarditis, including acute pericarditis and healed pericarditis,and rheumatoid heart disease; neoplastic heart disease, including butnot limited to, primary cardiac tumors, such as myxoma, lipoma,papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac effectsof noncardiac neoplasms; congenital heart disease, including but notlimited to, left-to-right shunts—late cyanosis, such as atrial septaldefect, ventricular septal defect, patent ductus arteriosus, andatrioventricular septal defect, right-to-left shunts—early cyanosis,such as tetralogy of fallot, transposition of great arteries, truncusarteriosus, tricuspid atresia, and total anomalous pulmonary venousconnection, obstructive congenital anomalies, such as coarctation ofaorta, pulmonary stenosis and atresia, and aortic stenosis and atresia,and disorders involving cardiac transplantation.

Disorders involving blood vessels include, but are not limited to,responses of vascular cell walls to injury, such as endothelialdysfunction and endothelial activation and intimal thickening; vasculardiseases including, but not limited to, congenital anomalies, such asarteriovenous fistula, atherosclerosis, and hypertensive vasculardisease, such as hypertension; inflammatory disease—the vasculitides,such as giant cell (temporal) arteritis, Takayasu arteritis,polyarteritis nodosa (classic), Kawasaki syndrome (mucocutaneous lymphnode syndrome), microscopic polyanglitis (microscopic polyarteritis,hypersensitivity or leukocytoclastic anglitis), Wegener granulomatosis,thromboanglitis obliterans (Buerger disease), vasculitis associated withother disorders, and infectious arteritis; Raynaud disease; aneurysmsand dissection, such as abdominal aortic aneurysms, syphilitic (luetic)aneurysms, and aortic dissection (dissecting hematoma); disorders ofveins and lymphatics, such as varicose veins, thrombophlebitis andphlebothrombosis, obstruction of superior vena cava (superior vena cavasyndrome), obstruction of inferior vena cava (inferior vena cavasyndrome), and lymphangitis and lymphedema; tumors, including benigntumors and tumor-like conditions, such as hemangioma, lymphangioma,glomus tumor (glomangioma), vascular ectasias, and bacillaryangiomatosis, and intermediate-grade (borderline low-grade malignant)tumors, such as Kaposi sarcoma and hemangloendothelioma, and malignanttumors, such as angiosarcoma and hemangiopericytoma; and pathology oftherapeutic interventions in vascular disease, such as balloonangioplasty and related techniques and vascular replacement, such ascoronary artery bypass graft surgery.

Disorders involving red cells include, but are not limited to, anemias,such as hemolytic anemias, including hereditary spherocytosis, hemolyticdisease due to erythrocyte enzyme defects: glucose-6-phosphatedehydrogenase deficiency, sickle cell disease, thalassemia syndromes,paroxysmal nocturnal hemoglobinuria, immunohemolytic anemia, andhemolytic anemia resulting from trauma to red cells; and anemias ofdiminished erythropoiesis, including megaloblastic anemias, such asanemias of vitamin B₁₂ deficiency: pernicious anemia, and anemia offolate deficiency, iron deficiency anemia, anemia of chronic disease,aplastic anemia, pure red cell aplasia, and other forms of marrowfailure.

Disorders involving the thymus include developmental disorders, such asDiGeorge syndrome with thymic hypoplasia or aplasia; thymic cysts;thymic hypoplasia, which involves the appearance of lymphoid follicleswithin the thymus, creating thymic follicular hyperplasia; and thymomas,including germ cell tumors, lynphomas, Hodgkin disease, and carcinoids.Thymomas can include benign or encapsulated thymoma, and malignantthymoma Type I (invasive thymoma) or Type II, designated thymiccarcinoma.

Disorders involving B-cells include, but are not limited to precursorB-cell neoplasms, such as lymphoblastic leukemia/lymphoma. PeripheralB-cell neoplasms include, but are not limited to, chronic lymphocyticleukemia/small lymphocytic lymphoma, follicular lymphoma, diffuse largeB-cell lymphoma, Burkitt lymphoma, plasma cell neoplasms, multiplemyeloma, and related entities, lymphoplasmacytic lymphoma (Waldenstrommacroglobulinemia), mantle cell lymphoma, marginal zone lymphoma(MALToma), and hairy cell leukemia.

Disorders involving the kidney include, but are not limited to,congenital anomalies including, but not limited to, cystic diseases ofthe kidney, that include but are not limited to, cystic renal dysplasia,autosomal dominant (adult) polycystic kidney disease, autosomalrecessive (childhood) polycystic kidney disease, and cystic diseases ofrenal medulla, which include, but are not limited to, medullary spongekidney, and nephronophthisis-uremic medullary cystic disease complex,acquired (dialysis-associated) cystic disease, such as simple cysts;glomerular diseases including pathologies of glomerular injury thatinclude, but are not limited to, in situ immune complex deposition, thatincludes, but is not limited to, anti-GBM nephritis, Heymann nephritis,and antibodies against planted antigens, circulating immune complexnephritis, antibodies to glomerular cells, cell-mediated immunity inglomerulonephritis, activation of alternative complement pathway,epithelial cell injury, and pathologies involving mediators ofglomerular injury including cellular and soluble mediators, acuteglomerulonephritis, such as acute proliferative (poststreptococcal,postinfectious) glomerulonephritis, including but not limited to,poststreptococcal glomerulonephritis and nonstreptococcal acuteglomerulonephritis, rapidly progressive (crescentic) glomerulonephritis,nephrotic syndrome, membranous glomerulonephritis (membranousnephropathy), minimal change disease (lipoid nephrosis), focal segmentalglomerulosclerosis, membranoproliferative glomerulonephritis, IgAnephropathy (Berger disease), focal proliferative and necrotizingglomerulonephritis (focal glomerulonephritis), hereditary nephritis,including but not limited to, Alport syndrome and thin membrane disease(benign familial hematuria), chronic glomerulonephritis, glomerularlesions associated with systemic disease, including but not limited to,systemic lupus erythematosus, Henoch-Schonlein purpura, bacterialendocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary andimmunotactoid glomerulonephritis, and other systemic disorders; diseasesaffecting tubules and interstitium, including acute tubular necrosis andtubulointerstitial nephritis, including but not limited to,pyelonephritis and urinary tract infection, acute pyelonephritis,chronic pyelonephritis and reflux nephropathy, and tubulointerstitialnephritis induced by drugs and toxins, including but not limited to,acute drug-induced interstitial nephritis, analgesic abuse nephropathy,nephropathy associated with nonsteroidal anti-inflammatory drugs, andother tubulointerstitial diseases including, but not limited to, uratenephropathy, hypercalcemia and nephrocalcinosis, and multiple myeloma;diseases of blood vessels including benign nephrosclerosis, malignanthypertension and accelerated nephrosclerosis, renal artery stenosis, andthrombotic microangiopathies including, but not limited to, classic(childhood) hemolytic-uremic syndrome, adult hemolytic-uremicsyndrome/thrombotic thrombocytopenic purpura, idiopathic HUS/TTP, andother vascular disorders including, but not limited to, atheroscleroticischemic renal disease, atheroembolic renal disease, sickle cell diseasenephropathy, diffuse cortical necrosis, and renal infarcts; urinarytract obstruction (obstructive uropathy); urolithiasis (renal calculi,stones); and tumors of the kidney including, but not limited to, benigntumors, such as renal papillary adenoma, renal fibroma or hamartoma(renomedullary interstitial cell tumor), angiomyolipoma, and oncocytoma,and malignant tumors, including renal cell carcinoma (hypernephroma,adenocarcinoma of kidney), which includes urothelial carcinomas of renalpelvis.

Disorders of the breast include, but are not limited to, disorders ofdevelopment; inflammations, including but not limited to, acutemastitis, periductal mastitis, periductal mastitis (recurrent subareolarabscess, squamous metaplasia of lactiferous ducts), mammary ductectasia, fat necrosis, granulomatous mastitis, and pathologiesassociated with silicone breast implants; fibrocystic changes;proliferative breast disease including, but not limited to, epithelialhyperplasia, sclerosing adenosis, and small duct papillomas; tumorsincluding, but not limited to, stromal tumors such as fibroadenoma,phyllodes tumor, and sarcomas, and epithelial tumors such as large ductpapilloma; carcinoma of the breast including in situ (noninvasive)carcinoma that includes ductal carcinoma in situ (including Paget'sdisease) and lobular carcinoma in situ, and invasive (infiltrating)carcinoma including, but not limited to, invasive ductal carcinoma, nospecial type, invasive lobular carcinoma, medullary carcinoma, colloid(mucinous) carcinoma, tubular carcinoma, and invasive papillarycarcinoma, and miscellaneous malignant neoplasms.

Disorders in the male breast include, but are not limited to,gynecomastia and carcinoma.

Disorders involving the testis and epididymis include, but are notlimited to, congenital anomalies such as cryptorchidism, regressivechanges such as atrophy, inflammations such as nonspecific epididymitisand orchitis, granulomatous (autoimmune) orchitis, and specificinflammations including, but not limited to, gonorrhea, mumps,tuberculosis, and syphilis, vascular disturbances including torsion,testicular tumors including germ cell tumors that include, but are notlimited to, seminoma, spermatocytic seminoma, embryonal carcinoma, yolksac tumor choriocarcinoma, teratoma, and mixed tumors, tumore of sexcord-gonadal stroma including, but not limited to, Leydig (interstitial)cell tumors and sertoli cell tumors (androblastoma), and testicularlymphoma, and miscellaneous lesions of tunica vaginalis.

Disorders involving the prostate include, but are not limited to,inflammations, benign enlargement, for example, nodular hyperplasia(benign prostatic hypertrophy or hyperplasia), and tumors such ascarcinoma.

Disorders involving the thyroid include, but are not limited to,hyperthyroidism; hypothyroidism including, but not limited to, cretinismand myxedema; thyroiditis including, but not limited to, hashimotothyroiditis, subacute (granulomatous) thyroiditis, and subacutelymphocytic (painless) thyroiditis; Graves disease; diffuse andmultinodular goiter including, but not limited to, diffuse nontoxic(simple) goiter and multinodular goiter; neoplasms of the thyroidincluding, but not limited to, adenomas, other benign tumors, andcarcinomas, which include, but are not limited to, papillary carcinoma,follicular carcinoma, medullary carcinoma, and anaplastic carcinoma; andcogenital anomalies.

Disorders involving the skeletal muscle include tumors such asrhabdomyosarcoma.

Disorders involving the pancreas include those of the exocrine pancreassuch as congenital anomalies, including but not limited to, ectopicpancreas; pancreatitis, including but not limited to, acutepancreatitis; cysts, including but not limited to, pseudocysts; tumors,including but not limited to, cystic tumors and carcinoma of thepancreas; and disorders of the endocrine pancreas such as, diabetesmellitus; islet cell tumors, including but not limited to, insulinomas,gastrinomas, and other rare islet cell tumors.

Disorders involving the small intestine include the malabsorptionsyndromes such as, celiac sprue, tropical sprue (postinfectious sprue),whipple disease, disaccharidase (lactase) deficiency,abetalipoproteinemia, and tumors of the small intestine includingadenomas and adenocarcinoma.

Disorders related to reduced platelet number, thrombocytopenia, includeidiopathic thrombocytopenic purpura, including acute idiopathicthrombocytopenic purpura, drug-induced thrombocytopenia, HIV-associatedthrombocytopenia, and thrombotic microangiopathies: thromboticthrombocytopenic purpura and hemolytic-uremic syndrome.

Disorders involving precursor T-cell neoplasms include precursor Tlymphoblastic leukemia/lymphoma. Disorders involving peripheral T-celland natural killer cell neoplasms include T-cell chronic lymphocyticleukemia, large granular lymphocytic leukemia, mycosis fungoides andSézary syndrome, peripheral T-cell lymphoma, unspecified,angioimmunoblastic T-cell lymphoma, angiocentric lymphoma (NK/T-celllymphoma4a), intestinal T-cell lymphoma, adult T-cell leukemia/lymphoma,and anaplastic large cell lymphoma.

Disorders involving the ovary include, for example, polycystic ovariandisease, Stein-leventhal syndrome, Pseudomyxoma peritonei and stromalhyperthecosis; ovarian tumors such as, tumors of coelomic epithelium,serous tumors, mucinous tumors, endometeriod tumors, clear celladenocarcinoma, cystadenofibroma, brenner tumor, surface epithelialtumors; germ cell tumors such as mature (benign) teratomas, monodermalteratomas, immature malignant teratomas, dysgerminoma, endodermal sinustumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-thecacell tumors, the coma-fibromas, and roblastomas, hill cell tumors, andgonadoblastoma; and metastatic tumors such as Krukenberg tumors.

Bone-forming cells include the osteoprogenitor cells, osteoblasts, andosteocytes. The disorders of the bone are complex because they may havean impact on the skeleton during any of its stages of development.Hence, the disorders may have variable manifestations and may involveone, multiple or all bones of the body. Such disorders include,congenital malformations, achondroplasia and thanatophoric dwarfism,diseases associated with abnormal matix such as type 1 collagen disease,osteoporosis, Paget disease, rickets, osteomalacia, high-turnoverosteodystrophy, low-turnover of aplastic disease, osteonecrosis,pyogenic osteomyelitis, tuberculous osteomyelitism, osteoma, osteoidosteoma, osteoblastoma, osteosarcoma, osteochondroma, chondromas,chondroblastoma, chondromyxoid fibroma, chondrosarcoma, fibrous corticaldefects, fibrous dysplasia, fibrosarcoma, malignant fibroushistiocytoma, Ewing sarcoma, primitive neuroectodermal tumor, giant celltumor, and metastatic tumors.

A novel human phosphatidylserine synthase gene sequence, referred to as32670, is provided. This gene sequence and variants and fragmentsthereof are encompassed by the term “phosphatidylserine synthase-like”molecules or sequences as used herein. The phosphatidylserinesynthase-like sequences find use in modulating a phosphatidylserinesynthase function. By “modulating” is intended the upregulating ordownregulating of a response. That is, the compositions of the inventionaffect the targeted activity in either a positive or negative fashion.

The human phosphatidylserine synthase-like gene, clone 32670 wasidentified in a human osteoblast cDNA library. Clone 32670 encodes anmRNA transcript having the corresponding cDNA set forth in SEQ ID NO:5.This transcript has a 1461 nucleotide open reading frame (nucleotides14-1474 of SEQ ID NO:5; SEQ ID NO:7), which encodes a 487 amino acidprotein (SEQ ID NO:6).

In one embodiment, a 32670 protein includes at least one transmembranedomain. As used herein, the term “transmembrane domain” includes anamino acid sequence of about 17-22 amino acid residues in length thatspans a phospholipid membrane. Transmembrane domains are rich inhydrophobic residues, and typically have an α-helical structure. In apreferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more ofthe amino acids of a transmembrane domain are hydrophobic, e.g.,leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domainsare described in, for example,http://pfam.wustl.edu/cgi-bin/getdesc?name=7tm-1, and Zagotta et al.(1996) Annual Rev. Neuronsci. 19:235-63, the contents of which areincorporated herein by reference.

In a preferred embodiment, a 32670 polypeptide or protein has at leastone transmembrane domain or a region which includes at least 17, 18, 19,20, 21, or 22 amino acid residues and has at least about 60%, 70% 80%90% 95%, 99%, or 100% sequence identity with a “transmembrane domain,”e.g., at least one transmembrane domain of human 32670 (e.g., amino acidresidues 66-82, 96-112, 127-144, 246-262, 314-334, 345-362, 382-399, or410-420 of SEQ ID NO:6).

In one embodiment, a 32670 protein includes at least one“non-transmembrane domain.” As used herein, “non-transmembrane domains”are domains that reside outside of the membrane. When referring toplasma membranes, non-transmembrane domains include extracellulardomains (i.e., outside of the cell) and intracellular domains (i.e.,within the cell). When referring to membrane-bound proteins found inintracellular organelles (e.g., mitochondria, endoplasmic reticulum,peroxisomes and microsomes), non-transmembrane domains include thosedomains of the protein that reside in the cytosol (i.e., the cytoplasm),the lumen of the organelle, or the matrix or the intermembrane space(the latter two relate specifically to mitochondria organelles). TheC-terminal amino acid residue of a non-transmembrane domain is adjacentto an N-terminal amino acid residue of a transmembrane domain in anaturally occurring 32670, or 32670-like protein.

In a preferred embodiment, a 32670 polypeptide or protein has a“non-transmembrane domain” or a region which includes at least about1-200, preferably about 5-150, more preferably about 10-105 amino acids,and has at least about 60%, 70% 80% 90% 95%, 99% or 100% sequenceidentity with a “non-transmembrane domain”, e.g., a non-transmembranedomain of human 32670 (e.g., residues 1-65, 83-95, 113-126, 145-245,263-313, 335-344, 363-381, 400-409, or 430-487 of SEQ ID NO:6).Preferably, a non-transmembrane domain is capable of catalytic activity(e.g., phosphatidylserine synthase activity).

A non-transmembrane domain located at the N-terminus of a 32670 proteinor polypeptide is referred to herein as an “N-terminal non-transmembranedomain.” As used herein, an “N-terminal non-transmembrane domain”includes an amino acid sequence having about 1-120, 25-105, orpreferably 45-85 amino acid residues in length and is located outsidethe boundaries of a membrane. For example, an N-terminalnon-transmembrane domain is located at about amino acid residues 1-65 ofSEQ ID NO:6.

Similarly, a non-transmembrane domain located at the C-terminus of a32670 protein or polypeptide is referred to herein as a “C-terminalnon-transmembrane domain.” As used herein, an “C-terminalnon-transmembrane domain” includes an amino acid sequence having about1-120, preferably about 20-100, more preferably about 40-80 amino acidresidues in length and is located outside the boundaries of a membrane.For example, an C-terminal non-transmembrane domain is located at aboutamino acid residues 430-487 of SEQ ID NO:6.

A 32670 polypeptide can further include a signal sequence. As usedherein, a “signal sequence” refers to a peptide of about 5-70 amino acidresidues in length which occurs at the N-terminus of secretory andintegral membrane proteins and which contains a majority of hydrophobicamino acid residues. For example, a signal sequence contains at leastabout 5-60 amino acid residues, preferably about 6-20 amino acidresidues, more preferably about 7 amino acid residues, and has at leastabout 40-70%, preferably about 50-65%, and more preferably about 55-60%hydrophobic amino acid residues (e.g., alanine, valine, leucine,isoleucine, phenylalanine, tyrosine, tryptophan, or proline). Such a“signal sequence”, also referred to in the art as a “signal peptide”,serves to direct a protein containing such a sequence to a lipidbilayer. For example, in one embodiment, a 32670 protein contains asignal sequence of about amino acids 1-7 of SEQ ID NO:6. The “signalsequence” is cleaved during processing of the mature protein. The mature32670 protein corresponds to amino acids 8-487 of SEQ ID NO:6.

Prosite program analysis was used to predict various consensus siteswithin the 32670 protein. N-glycosylation sites were predicted at aa181-184 and 237-240. A cAMP- and cGMP-dependent protein kinasephosphorylation site was predicted at aa 44-47. Protein kinase Cphosphorylation sites were predicted at aa 93-95, 269-271, 272-274,283-285, 310-312, and 437-439. Casein kinase II phosphorylation siteswere predicted at aa 24-27, 47-50, 80-83, 85-88, 146-149, and 389-392. Atyrosine kinase phosphorylation site was predicted at aa 44-52.N-myristoylation sites were predicted at aa 96-101, 108-113, 257-262,419-424, 455-460, and 476-481. An amidation site was predicted at aa42-45. The human phosphatidylserine synthase-like protein possesses atype III leader peptidase domain from aa 314-338 as predicted by Mer,Version 2.

The 32670 protein shares approximately 85% identity with thephosphatidylserine synthase II from Cricetulus griseus and approximately86% identity with the murine phosphatidylserine synthase-2 as determinedby pairwise alignment (FIG. 13).

The 32670 displays approximately 52% identity from aa 45-325 andapproximately 33% identity from aa 305-464 to Prodom consensus sequencesfound in phosphatidylserine synthase I from Cricetulus longicaudatus(ProDom Accession No. PDO11831). H32670 also shares approximately 40%identity from aa 446 to a Prodom consensus sequence found in HepatocyteNuclear Factor 3 Forkhead Homolog 1 (HFH-1) from Rattus norvegicus andin the murine Fork Head Transcription Factor (Pro Dom Accession No.PD31440). Phosphatidylserine synthase I is a serine exchange enzyme (EC2.7.8.8) involved in the synthesis of phosphatidylserine.Phosphatidylserine synthase I functions in a base-exchange between freeL-serine and the polar head groups of pre-existing phospholipids. It canutilize phosphatidylcholine as a phosphatidyl donor. It also catalyzesthe choline and ethanolamine base-exchange reactions. See, for example,Kuge et al. (1991) J. Biol. Chem. 266:24184-24189. The HFH-1 and theFork Head Transcription Factor contain a conserved “fork head” domaininvolved in DNA-binding. See, for example, Haecker et al. (1995) EMBO J.14:5306-5317, Clevidence et al. (1993) Proc. Natl. Acad. Sci.90:3948-3952.

Preferred human phosphatidylserine synthase-like polypeptides of thepresent invention have an amino acid sequence sufficiently identical tothe amino acid sequence of SEQ ID NO:6. The term “sufficientlyidentical” is used herein to refer to a first amino acid or nucleotidesequence that contains a sufficient or minimum number of identical orequivalent (e.g., with a similar side chain) amino acid residues ornucleotides to a second amino acid or nucleotide sequence such that thefirst and second amino acid or nucleotide sequences have a commonstructural domain and/or common functional activity. For example, aminoacid or nucleotide sequences that contain a common structural domainhaving at least about 60% identity, preferably 65% identity, morepreferably 70%, 75%, or 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more identity are defined herein as sufficientlyidentical.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, 100% of the length of thereference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (1970)J. Mol. Biol. 48:444-453 algorithm which has been incorporated into theGAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused if the practitioner is uncertain about what parameters should beapplied to determine if a molecule is within a sequence identity orhomology limitation of the invention) is using a Blossum 62 scoringmatrix with a gap open penalty of 12, a gap extend penalty of 4, and aframeshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of E. Meyers and W. Miller (1989)CABIOS 4:11-17 which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to 32670 nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to 32670 protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al. (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See http://www.ncbi.nlm.nih.gov.

Accordingly, another embodiment of the invention features isolated humanphosphatidylserine synthase-like proteins and polypeptides having ahuman phosphatidylserine synthase-like protein activity. As usedinterchangeably herein, a “human phosphatidylserine synthase-likeprotein activity”, “biological activity of a human phosphatidylserinesynthase-like protein”, or “functional activity of a humanphosphatidylserine synthase-like protein” refers to an activity exertedby a human phosphatidylserine synthase-like protein, polypeptide, ornucleic acid molecule on a human phosphatidylserine synthase-likeresponsive cell as determined in vivo, or in vitro, according tostandard assay techniques. A human phosphatidylserine synthase-likeactivity can be a direct activity, such as an association with or anenzymatic activity on a second protein, or an indirect activity, such asa cellular signaling activity mediated by interaction of the humanphosphatidylserine synthase-like protein with a second protein. In apreferred embodiment, human phosphatidylserine synthase-like activityincludes at least one or more modulating activities which may includestimulating and/or enhancing or inhibiting phosphatidylserine synthesis.

An “isolated” or “purified” human phosphatidylserine synthase-likenucleic acid molecule or protein, or biologically active portionthereof, is substantially free of other cellular material, or culturemedium when produced by recombinant techniques, or substantially free ofchemical precursors or other chemicals when chemically synthesized.Preferably, an “isolated” nucleic acid is free of sequences (preferablyprotein encoding sequences) that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forpurposes of the invention, “isolated” when used to refer to nucleic acidmolecules excludes isolated chromosomes. For example, in variousembodiments, the isolated human phosphatidylserine synthase-like nucleicacid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank thenucleic acid molecule in genomic DNA of the cell from which the nucleicacid is derived. A human phosphatidylserine synthase-like protein thatis substantially free of cellular material includes preparations ofhuman phosphatidylserine synthase-like protein having less than about30%, 20%, 10%, or 5% (by dry weight) of non-human phosphatidylserinesynthase-like protein (also referred to herein as a “contaminatingprotein”). When the human phosphatidylserine synthase-like protein orbiologically active portion thereof is recombinantly produced,preferably, culture medium represents less than about 30%, 20%, 10%, or5% of the volume of the protein preparation. When humanphosphatidylserine synthase-like protein is produced by chemicalsynthesis, preferably the protein preparations have less than about 30%,20%, 10%, or 5% (by dry weight) of chemical precursors or non-humanphosphatidylserine synthase-like chemicals.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculescomprising nucleotide sequences encoding human phosphatidylserinesynthase-like proteins and polypeptides or biologically active portionsthereof, as well as nucleic acid molecules sufficient for use ashybridization probes to identify human phosphatidylserine synthase-likeencoding nucleic acids (e.g., human phosphatidylserine synthase-likemRNA) and fragments for use as PCR primers for the amplification ormutation of human phosphatidylserine synthase-like nucleic acidmolecules. As used herein, the term “nucleic acid molecule” is intendedto include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules(e.g., mRNA) and analogs of the DNA or RNA generated using nucleotideanalogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

Nucleotide sequences encoding the human phosphatidylserine synthase-likeproteins of the present invention include sequences set forth in SEQ IDNO:5, SEQ ID NO:7, and complements thereof. By “complement” is intendeda nucleotide sequence that is sufficiently complementary to a givennucleotide sequence such that it can hybridize to the given nucleotidesequence to thereby form a stable duplex. The corresponding amino acidsequence for the human phosphatidylserine synthase-like protein encodedby these nucleotide sequences is set forth in SEQ ID NO:6. The inventionalso encompasses nucleic acid molecules comprising nucleotide sequencesencoding partial-length human phosphatidylserine synthase-like proteins,including the sequence set forth in SEQ ID NO:6, and complementsthereof.

Nucleic acid molecules that are fragments of these humanphosphatidylserine synthase-like nucleotide sequences are alsoencompassed by the present invention. By “fragment” is intended aportion of the nucleotide sequence encoding a human phosphatidylserinesynthase-like protein. A fragment of a human phosphatidylserinesynthase-like nucleotide sequence may encode a biologically activeportion of a human phosphatidylserine synthase-like protein, or it maybe a fragment that can be used as a hybridization probe or PCR primerusing methods disclosed below. A biologically active portion of a humanphosphatidylserine synthase-like protein can be prepared by isolating aportion of one of the human phosphatidylserine synthase-like nucleotidesequences of the invention, expressing the encoded portion of the humanphosphatidylserine synthase-like protein (e.g., by recombinantexpression in vitro), and assessing the activity of the encoded portionof the human phosphatidylserine synthase-like protein. Nucleic acidmolecules that are fragments of a human phosphatidylserine synthase-likenucleotide sequence comprise at least 15, 20, 50, 75, 100, 200, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600,1650, 1700, 1750, 1800 nucleotides, or up to the number of nucleotidespresent in a full-length human phosphatidylserine synthase-likenucleotide sequence disclosed herein (for example, 1852 nucleotides forSEQ ID NO:5) depending upon the intended use.

Alternatively, a nucleic acid molecule that is a fragment of a humanphosphatidylserine synthase-like nucleotide sequence of the presentinvention comprises a nucleotide sequence consisting of nucleotides1-100, 101-200, 201-300, 301-400, 401-500, 501-600, 601-700, 701-800,801-900, 901-1000, 1001-1100, 1101-1200, 1201-1300, 1301-1400,1401-1500, 1501-1600, 1601-1700, 1701-1800, or 1801-1852 of SEQ ID NO:5.

It is understood that isolated fragments include any contiguous sequencenot disclosed prior to the invention as well as sequences that aresubstantially the same and which are not disclosed. Accordingly, if anisolated fragment is disclosed prior to the present invention, thatfragment is not intended to be encompassed by the invention. When asequence is not disclosed prior to the present invention, an isolatednucleic acid fragment is at least about 12, 15, 20, 25, or 30 contiguousnucleotides. Other regions of the nucleotide sequence may comprisefragments of various sizes, depending upon potential homology withpreviously disclosed sequences.

A fragment of a human phosphatidylserine synthase-like nucleotidesequence that encodes a biologically active portion of a humanphosphatidylserine synthase-like protein of the invention will encode atleast 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, or 300contiguous amino acids, or up to the total number of amino acids presentin a full-length human phosphatidylserine synthase-like protein of theinvention (for example, 487 amino acids for SEQ ID NO:6). Fragments of ahuman phosphatidylserine synthase-like nucleotide sequence that areuseful as hybridization probes for PCR primers generally need not encodea biologically active portion of a human phosphatidylserinesynthase-like protein.

Nucleic acid molecules that are variants of the human phosphatidylserinesynthase-like nucleotide sequences disclosed herein are also encompassedby the present invention. “Variants” of the human phosphatidylserinesynthase-like nucleotide sequences include those sequences that encodethe human phosphatidylserine synthase-like proteins disclosed herein butthat differ conservatively because of the degeneracy of the geneticcode. These naturally occurring allelic variants can be identified withthe use of well-known molecular biology techniques, such as polymerasechain reaction (PCR) and hybridization techniques as outlined below.Variant nucleotide sequences also include synthetically derivednucleotide sequences that have been generated, for example, by usingsite-directed mutagenesis but which still encode the humanphosphatidylserine synthase-like proteins disclosed in the presentinvention as discussed below. Generally, nucleotide sequence variants ofthe invention will have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a particularnucleotide sequence disclosed herein. A variant human phosphatidylserinesynthase-like nucleotide sequence will encode a human phosphatidylserinesynthase-like protein that has an amino acid sequence having at least45%, 55%, 65%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity to the amino acid sequence of a human phosphatidylserinesynthase-like protein disclosed herein.

In addition to the human phosphatidylserine synthase-like nucleotidesequences shown in SEQ ID NO:5 and SEQ ID NO:7, it will be appreciatedby those skilled in the art that DNA sequence polymorphisms that lead tochanges in the amino acid sequences of human phosphatidylserinesynthase-like proteins may exist within a population (e.g., the humanpopulation). Such genetic polymorphism in a human phosphatidylserinesynthase-like gene may exist among individuals within a population dueto natural allelic variation. An allele is one of a group of genes thatoccur alternatively at a given genetic locus. As used herein, the terms“gene” and “recombinant gene” refer to nucleic acid molecules comprisingan open reading frame encoding a human phosphatidylserine synthase-likeprotein, preferably a mammalia human phosphatidylserine synthase-likeprotein. As used herein, the phrase “allelic variant” refers to anucleotide sequence that occurs at a human phosphatidylserinesynthase-like locus or to a polypeptide encoded by the nucleotidesequence. Such natural allelic variations can typically result in 1-5%variance in the nucleotide sequence of the human phosphatidylserinesynthase-like gene. Any and all such nucleotide variations and resultingamino acid polymorphisms or variations in a human phosphatidylserinesynthase-like sequence that are the result of natural allelic variationand that do not alter the functional activity of humanphosphatidylserine synthase-like proteins are intended to be within thescope of the invention.

Moreover, nucleic acid molecules encoding human phosphatidylserinesynthase-like proteins from other species (human phosphatidylserinesynthase-like homologues), which have a nucleotide sequence differingfrom that of the human phosphatidylserine synthase-like sequencesdisclosed herein, are intended to be within the scope of the invention.For example, nucleic acid molecules corresponding to natural allelicvariants and homologues of the huma human phosphatidylserinesynthase-like cDNA of the invention can be isolated based on theiridentity to the huma human phosphatidylserine synthase-like nucleic aciddisclosed herein using the human cDNA, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions as disclosed below.

In addition to naturally-occurring allelic variants of the humanphosphatidylserine synthase-like sequences that may exist in thepopulation, the skilled artisan will further appreciate that changes canbe introduced by mutation into the nucleotide sequences of the inventionthereby leading to changes in the amino acid sequence of the encodedhuman phosphatidylserine synthase-like proteins, without altering thebiological activity of the human phosphatidylserine synthase-likeproteins. Thus, an isolated nucleic acid molecule encoding a humanphosphatidylserine synthase-like protein having a sequence that differsfrom that of SEQ ID NO:5 or SEQ ID NO:7 can be created by introducingone or more nucleotide substitutions, additions, or deletions into thecorresponding nucleotide sequence disclosed herein, such that one ormore amino acid substitutions, additions or deletions are introducedinto the encoded protein. Mutations can be introduced by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Such variant nucleotide sequences are also encompassed bythe present invention.

For example, preferably, conservative amino acid substitutions may bemade at one or more predicted, preferably nonessential amino acidresidues. A “nonessential” amino acid residue is a residue that can bealtered from the wild-type sequence of a human phosphatidylserinesynthase-like protein (e.g., the sequence of SEQ ID NO:6) withoutaltering the biological activity, whereas an “essential” amino acidresidue is required for biological activity. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

Alternatively, variant human phosphatidylserine synthase-like nucleotidesequences can be made by introducing mutations randomly along all orpart of a human phosphatidylserine synthase-like coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedfor human phosphatidylserine synthase-like biological activity toidentify mutants that retain activity. Following mutagenesis, theencoded protein can be expressed recombinantly, and the activity of theprotein can be determined using standard assay techniques.

Thus the nucleotide sequences of the invention include the sequencesdisclosed herein as well as fragments and variants thereof. The humanphosphatidylserine synthase-like nucleotide sequences of the invention,and fragments and variants thereof, can be used as probes and/or primersto identify and/or clone human phosphatidylserine synthase-likehomologues in other cell types, e.g., from other tissues, as well ashuman phosphatidylserine synthase-like homologues from other mammals.Such probes can be used to detect transcripts or genomic sequencesencoding the same or identical proteins. These probes can be used aspart of a diagnostic test kit for identifying cells or tissues thatmisexpress a human phosphatidylserine synthase-like protein, such as bymeasuring levels of a human phosphatidylserine synthase-like-encodingnucleic acid in a sample of cells from a subject, e.g., detecting humanphosphatidylserine synthase-like mRNA levels or determining whether agenomic human phosphatidylserine synthase-like gene has been mutated ordeleted.

In this manner, methods such as PCR, hybridization, and the like can beused to identify such sequences having substantial identity to thesequences of the invention. See, for example, Sambrook et al. (1989)Molecular Cloning: Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.) and Innis et al. (1990) PCRProtocols: A Guide to Methods and Applications (Academic Press, NY).Human phosphatidylserine synthase-like nucleotide sequences isolatedbased on their sequence identity to the human phosphatidylserinesynthase-like nucleotide sequences set forth herein or to fragments andvariants thereof are encompassed by the present invention.

In a hybridization method, all or part of a known humanphosphatidylserine synthase-like nucleotide sequence can be used toscreen cDNA or genomic libraries. Methods for construction of such cDNAand genomic libraries are generally known in the art and are disclosedin Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2ded., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). Theso-called hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments, or other oligonucleotides, and may be labeledwith a detectable group such as ³²P, or any other detectable marker,such as other radioisotopes, a fluorescent compound, an enzyme, or anenzyme co-factor. Probes for hybridization can be made by labelingsynthetic oligonucleotides based on the known human phosphatidylserinesynthase-like nucleotide sequence disclosed herein. Degenerate primersdesigned on the basis of conserved nucleotides or amino acid residues ina known human phosphatidylserine synthase-like nucleotide sequence orencoded amino acid sequence can additionally be used. The probetypically comprises a region of nucleotide sequence that hybridizesunder stringent conditions to at least about 12, preferably about 25,more preferably about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or400 consecutive nucleotides of a human phosphatidylserine synthase-likenucleotide sequence of the invention or a fragment or variant thereof.Preparation of probes for hybridization is generally known in the artand is disclosed in Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.), herein incorporated by reference.

For example, in one embodiment, a previously unidentified humanphosphatidylserine synthase-like nucleic acid molecule hybridizes understringent conditions to a probe that is a nucleic acid moleculecomprising one of the human phosphatidylserine synthase-like nucleotidesequences of the invention or a fragment thereof. In another embodiment,the previously unknown human phosphatidylserine synthase-like nucleicacid molecule is at least 300, 325, 350, 375, 400, 425, 450, 500, 550,600, 650, 700, 800, 900, 1000, 2,000, 3,000, 4,000 or 5,000 nucleotidesin length and hybridizes under stringent conditions to a probe that is anucleic acid molecule comprising one of the human phosphatidylserinesynthase-like nucleotide sequences disclosed herein or a fragmentthereof.

Accordingly, in another embodiment, an isolated previously unknown humanphosphatidylserine synthase-like nucleic acid molecule of the inventionis at least 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700,800, 900, 1000, 1,100, 1,200, 1,300, 1,400 nucleotides in length andhybridizes under stringent conditions to a probe that is a nucleic acidmolecule comprising one of the nucleotide sequences of the invention,preferably the coding sequence set forth in SEQ ID NO:5 (shown in SEQ IDNO:7) or a complement, fragment, or variant thereof.

As used herein, the term “hybridizes under stringent conditions”describes conditions for hybridization and washing. Stringent conditionsare known to those skilled in the art and can be found in CurrentProtocols in Molecular Biology John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. Aqueous and nonaqueous methods are described in thatreference and either can be used. A preferred, example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50° C. Another example of stringent hybridizationconditions are hybridization in 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at55° C. A further example of stringent hybridization conditions arehybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.Preferably, stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 65° C. Particularly preferredstringency conditions (and the conditions that should be used if thepractitioner is uncertain about what conditions should be applied todetermine if a molecule is within a hybridization limitation of theinvention) are 0.5M Sodium Phosphate, 7% SDS at 65° C., followed by oneor more washes at 0.2×SSC, 1% SDS at 65° C. Preferably, an isolatednucleic acid molecule of the invention that hybridizes under stringentconditions to the sequence of SEQ ID NO:5 or SEQ ID NO:7 corresponds toa naturally-occurring nucleic acid molecule.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein).

Thus, in addition to the human phosphatidylserine synthase-likenucleotide sequences disclosed herein and fragments and variantsthereof, the isolated nucleic acid molecules of the invention alsoencompass homologous DNA sequences identified and isolated from othercells and/or organisms by hybridization with entire or partial sequencesobtained from the human phosphatidylserine synthase-like nucleotidesequences disclosed herein or variants and fragments thereof.

The present invention also encompasses antisense nucleic acid molecules,i.e., molecules that are complementary to a sense nucleic acid encodinga protein, e.g., complementary to the coding strand of a double-strandedcDNA molecule, or complementary to an mRNA sequence. Accordingly, anantisense nucleic acid can hydrogen bond to a sense nucleic acid. Theantisense nucleic acid can be complementary to an entire humanphosphatidylserine synthase-like coding strand, or to only a portionthereof, e.g., all or part of the protein coding region (or open readingframe). An antisense nucleic acid molecule can be antisense to anoncoding region of the coding strand of a nucleotide sequence encodinga human phosphatidylserine synthase-like protein. The noncoding regionsare the 5′ and 3′ sequences that flank the coding region and are nottranslated into amino acids.

Given the coding-strand sequence encoding a human phosphatidylserinesynthase-like protein disclosed herein (e.g., SEQ ID NO:6), antisensenucleic acids of the invention can be designed according to the rules ofWatson and Crick base pairing. The antisense nucleic acid molecule canbe complementary to the entire coding region of human phosphatidylserinesynthase-like mRNA, but more preferably is an oligonucleotide that isantisense to only a portion of the coding or noncoding region of humanphosphatidylserine synthase-like mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of human phosphatidylserine synthase-like mRNA.An antisense oligonucleotide can be, for example, about 5, 10, 15, 20,25, 30, 35, 40, 45, or 50 nucleotides in length. An antisense nucleicacid of the invention can be constructed using chemical synthesis andenzymatic ligation procedures known in the art.

For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, including, but not limited to, for example e.g., phosphorothioatederivatives and acridine substituted nucleotides. Alternatively, theantisense nucleic acid can be produced biologically using an expressionvector into which a nucleic acid has been subcloned in an antisenseorientation (i.e., RNA transcribed from the inserted nucleic acid willbe of an antisense orientation to a target nucleic acid of interest,described further in the following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a humanphosphatidylserine synthase-like protein to thereby inhibit expressionof the protein, e.g., by inhibiting transcription and/or translation. Anexample of a route of administration of antisense nucleic acid moleculesof the invention includes direct injection at a tissue site.Alternatively, antisense nucleic acid molecules can be modified totarget selected cells and then administered systemically. For example,antisense molecules can be linked to peptides or antibodies to form acomplex that specifically binds to receptors or antigens expressed on aselected cell surface. The antisense nucleic acid molecules can also bedelivered to cells using the vectors described herein. To achievesufficient intracellular concentrations of the antisense molecules,vector constructs in which the antisense nucleic acid molecule is placedunder the control of a strong pol II or pol III promoter are preferred.

An antisense nucleic acid molecule of the invention can be an α-anomericnucleic acid molecule. An a-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

The invention also encompasses ribozymes, which are catalytic RNAmolecules with ribonuclease activity that are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Ribozymes (e.g., hammerhead ribozymes (describedin Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used tocatalytically cleave human phosphatidylserine synthase-like mRNAtranscripts to thereby inhibit translation of human phosphatidylserinesynthase-like mRNA. A ribozyme having specificity for a humanphosphatidylserine synthase-like-encoding nucleic acid can be designedbased upon the nucleotide sequence of a human phosphatidylserinesynthase-like cDNA disclosed herein (e.g., SEQ ID NO:5 or SEQ ID NO:7).See, e.g., Cech et al., U.S. Pat. No. 4,987,071; and Cech et al., U.S.Pat. No. 5,116,742. Alternatively, human phosphatidylserinesynthase-like mRNA can be used to select a catalytic RNA having aspecific ribonuclease activity from a pool of RNA molecules. See, e.g.,Bartel and Szostak (1993) Science 261:1411-1418.

The invention also encompasses nucleic acid molecules that form triplehelical structures. For example, human phosphatidylserine synthase-likegene expression can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the human phosphatidylserinesynthase-like protein (e.g., the human phosphatidylserine synthase-likepromoter and/or enhancers) to form triple helical structures thatprevent transcription of the human phosphatidylserine synthase-like genein target cells. See generally Helene (1991) Anticancer Drug Des.6(6):569; Helene (1992) Ann. N.Y. Acad. Sci. 660:27; and Maher (1992)Bioassays 14(12):807.

In preferred embodiments, the nucleic acid molecules of the inventioncan be modified at the base moiety, sugar moiety, or phosphate backboneto improve, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal. (1996) Bioorganic & Medicinal Chemistry 4:5). As used herein, theterms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics,e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid-phase peptide synthesis protocols as described in Hyrupet al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci.USA 93:14670.

PNAs of a human phosphatidylserine synthase-like molecule can be used intherapeutic and diagnostic applications. For example, PNAs can be usedas antisense or antigene agents for sequence-specific modulation of geneexpression by, e.g., inducing transcription or translation arrest orinhibiting replication. PNAs of the invention can also be used, e.g., inthe analysis of single base pair mutations in a gene by, e.g.,PNA-directed PCR clamping; as artificial restriction enzymes when usedin combination with other enzymes, e.g., S1 nucleases (Hyrup (1996),supra; or as probes or primers for DNA sequence and hybridization (Hyrup(1996), supra; Perry-O'Keefe et al. (1996), supra).

In another embodiment, PNAs of a human phosphatidylserine synthase-likemolecule can be modified, e.g., to enhance their stability, specificity,or cellular uptake, by attaching lipophilic or other helper groups toPNA, by the formation of PNA-DNA chimeras, or by the use of liposomes orother techniques of drug delivery known in the art. The synthesis ofPNA-DNA chimeras can be performed as described in Hyrup (1996), supra;Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63; Mag et al. (1989)Nucleic Acids Res. 17:5973; and Peterson et al. (1975) Bioorganic Med.Chem. Lett. 5:1119.

II. Isolated Human Phosphatidylserine Synthase-Like Proteins andAnti-Human Phosphatidylserine Synthase-Like Antibodies

Human phosphatidylserine synthase-like proteins are also encompassedwithin the present invention. By “human phosphatidylserine synthase-likeprotein” is intended a protein having the amino acid sequence set forthin SEQ ID NO: 2, as well as fragments, biologically active portions, andvariants thereof.

“Fragments” or “biologically active portions” include polypeptidefragments suitable for use as immunogens to raise anti-humanphosphatidylserine synthase-like antibodies. Fragments include peptidescomprising amino acid sequences sufficiently identical to or derivedfrom the amino acid sequence of a human phosphatidylserine synthase-likeprotein, or partial-length protein, of the invention and exhibiting atleast one activity of a human phosphatidylserine synthase-like protein,but which include fewer amino acids than the full-length (SEQ ID NO:6)human phosphatidylserine synthase-like protein disclosed herein.Typically, biologically active portions comprise a domain or motif withat least one activity of the human phosphatidylserine synthase-likeprotein. A biologically active portion of a human phosphatidylserinesynthase-like protein can be a polypeptide which is, for example, 10,25, 50, 100 or more amino acids in length. Such biologically activeportions can be prepared by recombinant techniques and evaluated for oneor more of the functional activities of a native humanphosphatidylserine synthase-like protein. As used here, a fragmentcomprises at least 5 contiguous amino acids of SEQ ID NO:6. Theinvention encompasses other fragments, however, such as any fragment inthe protein greater than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 16, 17, 18,19 or 20 amino acids.

By “variants” is intended proteins or polypeptides having an amino acidsequence that is at least about 60%, 65%, or 70%, preferably about 75%,85%, 90%, 91%, 923, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical tothe amino acid sequence of SEQ ID NO:6. Variants also includepolypeptides encoded by a nucleic acid molecule that hybridizes to thenucleic acid molecule of SEQ ID NO:5, SEQ ID NO:7, or a complementthereof, under stringent conditions. In another embodiment, a variant ofan isolated polypeptide of the present invention differs, by at least 1,but less than 5, 10, 20, 50, or 100 amino acid residues from thesequence shown in SEQ ID NO:6. If alignment is needed for thiscomparison the sequences should be aligned for maximum identity.“Looped” out sequences from deletions or insertions, or mismatches, areconsidered differences. Such variants generally retain the functionalactivity of the 32670 proteins of the invention. Variants includepolypeptides that differ in amino acid sequence due to natural allelicvariation or mutagenesis.

The invention also provides human phosphatidylserine synthase-likechimeric or fusion proteins. As used herein, a human phosphatidylserinesynthase-like “chimeric protein” or “fusion protein” comprises a humanphosphatidylserine synthase-like polypeptide operably linked to anon-human phosphatidylserine synthase-like polypeptide. A “humanphosphatidylserine synthase-like polypeptide” refers to a polypeptidehaving an amino acid sequence corresponding to a humanphosphatidylserine synthase-like protein, whereas a “non-humanphosphatidylserine synthase-like polypeptide” refers to a polypeptidehaving an amino acid sequence corresponding to a protein that is notsubstantially identical to the human phosphatidylserine synthase-likeprotein, e.g., a protein that is different from the humanphosphatidylserine synthase-like protein and which is derived from thesame or a different organism. Within a human phosphatidylserinesynthase-like fusion protein, the human phosphatidylserine synthase-likepolypeptide can correspond to all or a portion of a humanphosphatidylserine synthase-like protein, preferably at least onebiologically active portion of a human phosphatidylserine synthase-likeprotein. Within the fusion protein, the term “operably linked” isintended to indicate that the human phosphatidylserine synthase-likepolypeptide and the non-human phosphatidylserine synthase-likepolypeptide are fused in-frame to each other. The non-humanphosphatidylserine synthase-like polypeptide can be fused to theN-terminus or C-terminus of the human phosphatidylserine synthase-likepolypeptide.

One useful fusion protein is a GST-human phosphatidylserinesynthase-like fusion protein in which the human phosphatidylserinesynthase-like sequences are fused to the C-terminus of the GSTsequences. Such fusion proteins can facilitate the purification ofrecombinant human phosphatidylserine synthase-like proteins.

In yet another embodiment, the fusion protein is a humanphosphatidylserine synthase-like-immunoglobulin fusion protein in whichall or part of a human phosphatidylserine synthase-like protein is fusedto sequences derived from a member of the immunoglobulin protein family.The human phosphatidylserine synthase-like-immunoglobulin fusionproteins of the invention can be incorporated into pharmaceuticalcompositions and administered to a subject to inhibit an interactionbetween a human phosphatidylserine synthase-like ligand and a humanphosphatidylserine synthase-like protein on the surface of a cell,thereby suppressing human phosphatidylserine synthase-like-mediatedsignal transduction in vivo. The human phosphatidylserinesynthase-like-immunoglobulin fusion proteins can be used to affect thebioavailability of a human phosphatidylserine synthase-like cognateligand. Inhibition of the human phosphatidylserine synthase-likeligand/human phosphatidylserine synthase-like interaction may be usefultherapeutically. Moreover, the human phosphatidylserinesynthase-like-immunoglobulin fusion proteins of the invention can beused as immunogens to produce anti-human phosphatidylserinesynthase-like antibodies in a subject, to purify humanphosphatidylserine synthase-like ligands, and in screening assays toidentify molecules that inhibit the interaction of a humanphosphatidylserine synthase-like protein with a human phosphatidylserinesynthase-like ligand.

Preferably, a human phosphatidylserine synthase-like chimeric or fusionprotein of the invention is produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the differentpolypeptide sequences may be ligated together in-frame, or the fusiongene can be synthesized, such as with automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers that give rise to complementary overhangs betweentwo consecutive gene fragments, which can subsequently be annealed andreamplified to generate a chimeric gene sequence (see, e.g., Ausubel etal., eds. (1995) Current Protocols in Molecular Biology) (GreenePublishing and Wiley-Interscience, NY). Moreover, a humanphosphatidylserine synthase-like-encoding nucleic acid can be clonedinto a commercially available expression vector such that it is linkedin-frame to an existing fusion moiety. Variants of the humanphosphatidylserine synthase-like proteins can function as either humanphosphatidylserine synthase-like agonists (mimetics) or as humanphosphatidylserine synthase-like antagonists. Variants of the humanphosphatidylserine synthase-like protein can be generated bymutagenesis, e.g., discrete point mutation or truncation of the humanphosphatidylserine synthase-like protein. An agonist of the humanphosphatidylserine synthase-like protein can retain substantially thesame, or a subset, of the biological activities of the naturallyoccurring form of the human phosphatidylserine synthase-like protein. Anantagonist of the human phosphatidylserine synthase-like protein caninhibit one or more of the activities of the naturally occurring form ofthe human phosphatidylserine synthase-like protein by, for example,competitively binding to a downstream or upstream member of a cellularsignaling cascade that includes the human phosphatidylserinesynthase-like protein. Thus, specific biological effects can be elicitedby treatment with a variant of limited function. Treatment of a subjectwith a variant having a subset of the biological activities of thenaturally occurring form of the protein can have fewer side effects in asubject relative to treatment with the naturally occurring form of thehuman phosphatidylserine synthase-like proteins.

Variants of a human phosphatidylserine synthase-like protein thatfunction as either human phosphatidylserine synthase-like agonists or ashuman phosphatidylserine synthase-like antagonists can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutants,of a human phosphatidylserine synthase-like protein for humanphosphatidylserine synthase-like protein agonist or antagonist activity.In one embodiment, a variegated library of human phosphatidylserinesynthase-like variants is generated by combinatorial mutagenesis at thenucleic acid level and is encoded by a variegated gene library. Avariegated library of human phosphatidylserine synthase-like variantscan be produced by, for example, enzymatically ligating a mixture ofsynthetic oligonucleotides into gene sequences such that a degenerateset of potential human phosphatidylserine synthase-like sequences isexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g., for phage display) containing the set ofhuman phosphatidylserine synthase-like sequences therein. There are avariety of methods that can be used to produce libraries of potentialhuman phosphatidylserine synthase-like variants from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be performed in an automatic DNA synthesizer, and thesynthetic gene then ligated into an appropriate expression vector. Useof a degenerate set of genes allows for the provision, in one mixture,of all of the sequences encoding the desired set of potential humanphosphatidylserine synthase-like sequences. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang(1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem.53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)Nucleic Acid Res. 11:477).

In addition, libraries of fragments of a human phosphatidylserinesynthase-like protein coding sequence can be used to generate avariegated population of human phosphatidylserine synthase-likefragments for screening and subsequent selection of variants of a humanphosphatidylserine synthase-like protein. In one embodiment, a libraryof coding sequence fragments can be generated by treating adouble-stranded PCR fragment of a human phosphatidylserine synthase-likecoding sequence with a nuclease under conditions wherein nicking occursonly about once per molecule, denaturing the double-stranded DNA,renaturing the DNA to form double-stranded DNA which can includesense/antisense pairs from different nicked products, removingsingle-stranded portions from reformed duplexes by treatment with S1nuclease, and ligating the resulting fragment library into an expressionvector. By this method, one can derive an expression library thatencodes N-terminal and internal fragments of various sizes of the humanphosphatidylserine synthase-like protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of human phosphatidylserinesynthase-like proteins. The most widely used techniques, which areamenable to high through-put analysis, for screening large genelibraries typically include cloning the gene library into replicableexpression vectors, transforming appropriate cells with the resultinglibrary of vectors, and expressing the combinatorial genes underconditions in which detection of a desired activity facilitatesisolation of the vector encoding the gene whose product was detected.Recursive ensemble mutagenesis (REM), a technique that enhances thefrequency of functional mutants in the libraries, can be used incombination with the screening assays to identify humanphosphatidylserine synthase-like variants (Arkin and Yourvan (1992)Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) ProteinEngineering 6(3):327-331).

An isolated human phosphatidylserine synthase-like polypeptide of theinvention can be used as an immunogen to generate antibodies that bindhuman phosphatidylserine synthase-like proteins using standardtechniques for polyclonal and monoclonal antibody preparation. Thefull-length human phosphatidylserine synthase-like protein can be usedor, alternatively, the invention provides antigenic peptide fragments ofhuman phosphatidylserine synthase-like proteins for use as immunogens.The antigenic peptide of a human phosphatidylserine synthase-likeprotein comprises at least 8, preferably 10, 15, 20, or 30 amino acidresidues of the amino acid sequence shown in SEQ ID NO:6 and encompassesan epitope of a human phosphatidylserine synthase-like protein such thatan antibody raised against the peptide forms a specific immune complexwith the human phosphatidylserine synthase-like protein. Preferredepitopes encompassed by the antigenic peptide are regions of a humanphosphatidylserine synthase-like protein that are located on the surfaceof the protein, e.g., hydrophilic regions.

Accordingly, another aspect of the invention pertains to anti-humanphosphatidylserine synthase-like polyclonal and monoclonal antibodiesthat bind a human phosphatidylserine synthase-like protein. Polyclonalanti-human phosphatidylserine synthase-like antibodies can be preparedby immunizing a suitable subject (e.g., rabbit, goat, mouse, or othermammal) with a human phosphatidylserine synthase-like immunogen. Theanti-human phosphatidylserine synthase-like antibody titer in theimmunized subject can be monitored over time by standard techniques,such as with an enzyme linked immunosorbent assay (ELISA) usingimmobilized human phosphatidylserine synthase-like protein. At anappropriate time after immunization, e.g., when the anti-humanphosphatidylserine synthase-like antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497, the human B cell hybridoma technique (Kozbor et al.(1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al.(1985) in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld andSell (Alan R. Liss, Inc., New York, N.Y.), pp. 77-96) or triomatechniques. The technology for producing hybridomas is well known (seegenerally Coligan et al., eds. (1994) Current Protocols in Immunology(John Wiley & Sons, Inc., New York, N.Y.); Galfre et al. (1977) Nature266:550-52; Kenneth (1980) in Monoclonal Antibodies: A New Dimension InBiological Analyses (Plenum Publishing Corp., NY; and Lerner (1981) YaleJ. Biol. Med., 54:387-402).

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-human phosphatidylserine synthase-like antibody can beidentified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) with ahuman phosphatidylserine synthase-like protein to thereby isolateimmunoglobulin library members that bind the human phosphatidylserinesynthase-like protein. Kits for generating and screening phage displaylibraries are commercially available (e.g., the Pharmacia RecombinantPhage Antibody System, Catalog No. 27-9400-01; and the StratageneSurfZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examplesof methods and reagents particularly amenable for use in generating andscreening antibody display library can be found in, for example, U.S.Pat. No. 5,223,409; PCT Publication Nos. WO 92/18619; WO 91/17271; WO92/20791; WO 92/15679; 93/01288; WO 92/01047; 92/09690; and 90/02809;Fuchs et al. (1991) Bio/Techniques 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffiths et al. (1993) EMBO J. 12:725-734.

Additionally, recombinant anti-human phosphatidylserine synthase-likeantibodies, such as chimeric and humanized monoclonal antibodies,comprising both human and nonhuman portions, which can be made usingstandard recombinant DNA techniques, are within the scope of theinvention. Such chimeric and humanized monoclonal antibodies can beproduced by recombinant DNA techniques known in the art, for exampleusing methods described in PCT Publication Nos. WO 86/101533 and WO87/02671; European Patent Application Nos. 184,187, 171, 496, 125,023,and 173,494; U.S. Pat. Nos. 4,816,567 and 5,225,539; European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986)Bio/Techniques 4:214; Jones et al. (1986) Nature 321:552-525; Verhoeyanet al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.141:4053-4060.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice that are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. See, for example, Lonberg and Huszar (1995)Int. Rev. Immunol. 13:65-93); and U.S. Pat. Nos. 5,625,126; 5,633,425;5,569,825; 5,661,016; and 5,545,806. In addition, companies such asAbgenix, Inc. (Freemont, Calif.), can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. This technology is described by Jespers etal. (1994) Bio/Technology 12:899-903).

An anti-human phosphatidylserine synthase-like antibody (e.g.,monoclonal antibody) can be used to isolate human phosphatidylserinesynthase-like proteins by standard techniques, such as affinitychromatography or immunoprecipitation. An anti-human phosphatidylserinesynthase-like antibody can facilitate the purification of natural humanphosphatidylserine synthase-like protein from cells and of recombinantlyproduced human phosphatidylserine synthase-like protein expressed inhost cells. Moreover, an anti-human phosphatidylserine synthase-likeantibody can be used to detect human phosphatidylserine synthase-likeprotein (e.g., in a cellular lysate or cell supernatant) in order toevaluate the abundance and pattern of expression of the humanphosphatidylserine synthase-like protein. Anti-human phosphatidylserinesynthase-like antibodies can be used diagnostically to monitor proteinlevels in tissue as part of a clinical testing procedure, e.g., to, forexample, determine the efficacy of a given treatment regimen. Detectioncan be facilitated by coupling the antibody to a detectable substance.Examples of detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, and radioactive materials. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S, or ³H.

Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). The conjugates of the invention canbe used for modifying a given biological response, the drug moiety isnot to be construed as limited to classical chemical therapeutic agents.For example, the drug moiety may be a protein or polypeptide possessinga desired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, alpha-interferon,beta-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies'84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a humanphosphatidylserine synthase-like protein (or a portion thereof).“Vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked, such as a “plasmid”, acircular double-stranded DNA loop into which additional DNA segments canbe ligated, or a viral vector, where additional DNA segments can beligated into the viral genome. The vectors are useful for autonomousreplication in a host cell or may be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome (e.g., nonepisomal mammalianvectors). Expression vectors are capable of directing the expression ofgenes to which they are operably linked. In general, expression vectorsof utility in recombinant DNA techniques are often in the form ofplasmids (vectors). However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses, and adeno-associatedviruses), that serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell. This means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, operably linked to the nucleicacid sequence to be expressed. “Operably linked” is intended to meanthat the nucleotide sequence of interest is linked to the regulatorysequence(s) in a manner that allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in ahost cell when the vector is introduced into the host cell). The term“regulatory sequence” is intended to include promoters, enhancers, andother expression control elements (e.g., polyadenylation signals). See,for example, Goeddel (1990) in Gene Expression Technology: Methods inEnzymology 185 (Academic Press, San Diego, Calif.). Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cell and those that direct expression ofthe nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., human phosphatidylserinesynthase-like proteins, mutant forms of human phosphatidylserinesynthase-like proteins, fusion proteins, etc.).

It is further recognized that the nucleic acid sequences of theinvention can be altered to contain codons, which are preferred, or nonpreferred, for a particular expression system. For example, the nucleicacid can be one in which at least one altered codon, and preferably atleast 10%, or 20% of the codons have been altered such that the sequenceis optimized for expression in E. coli, yeast, human, insect, or CHOcells. Methods for determining such codon usage are well known in theart.

The recombinant expression vectors of the invention can be designed forexpression of human phosphatidylserine synthase-like protein inprokaryotic or eukaryotic host cells. Expression of proteins inprokaryotes is most often carried out in E. coli with vectors containingconstitutive or inducible promoters directing the expression of eitherfusion or nonfusion proteins. Fusion vectors add a number of amino acidsto a protein encoded therein, usually to the amino terminus of therecombinant protein. Typical fusion expression vectors include pGEX(Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40), pMAL(New England Biolabs, Beverly, Mass.), and pRIT5 (Pharmacia, Piscataway,N.J.) which fuse glutathione S-transferase (GST), maltose E bindingprotein, or protein A, respectively, to the target recombinant protein.Examples of suitable inducible nonfusion E. coli expression vectorsinclude pTrc (Amann et al. (1988) Gene 69:301-315) and pET 11d (Studieret al. (1990) in Gene Expression Technology: Methods in Enzymology 185(Academic Press, San Diego, Calif.), pp. 60-89). Strategies to maximizerecombinant protein expression in E. coli can be found in Gottesman(1990) in Gene Expression Technology: Methods in Enzymology 185(Academic Press, CA), pp. 119-128 and Wada et al. (1992) Nucleic AcidsRes. 20:2111-2118. Target gene expression from the pTrc vector relies onhost RNA polymerase transcription from a hybrid trp-lac fusion promoter.

Suitable eukaryotic host cells include insect cells (examples ofBaculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39)); yeast cells (examples of vectorsfor expression in yeast S. cereivisiae include pYepSec1 (Baldari et al.(1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2(Invitrogen Corporation, San Diego, Calif.), and pPicZ (InvitrogenCorporation, San Diego, Calif.)); or mammalian cells (mammalianexpression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC(Kaufman et al. (1987) EMBO J. 6:187:195)). Suitable mammalian cellsinclude Chinese hamster ovary cells (CHO) or COS cells. In mammaliancells, the expression vector's control functions are often provided byviral regulatory elements. For example, commonly used promoters arederived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus40. For other suitable expression systems for both prokaryotic andeukaryotic cells, see chapters 16 and 17 of Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.). See, Goeddel (1990) in GeneExpression Technology: Methods in Enzymology 185 (Academic Press, SanDiego, Calif.). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell but are stillincluded within the scope of the term as used herein.

A “purified preparation of cells”, as used herein, refers to, in thecase of plant or animal cells, an in vitro preparation of cells and notan entire intact plant or animal. In the case of cultured cells ormicrobial cells, it consists of a preparation of at least 10% and morepreferably 50% of the subject cells.

In one embodiment, the expression vector is a recombinant mammalianexpression vector that comprises tissue-specific regulatory elementsthat direct expression of the nucleic acid preferentially in aparticular cell type. Suitable tissue-specific promoters include thealbumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev.1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv.Immunol. 43:235-275), in particular promoters of T cell receptors(Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins(Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell33:741-748), neuron-specific promoters (e.g., the neurofilamentpromoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science230:912-916), and mammary gland-specific promoters (e.g., milk wheypromoter; U.S. Pat. No. 4,873,316 and European Application PatentPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine homeobox (hox) promoters (Kessel andGruss (1990) Science 249:374-379), the a-fetoprotein promoter (Campesand Tilghman (1989) Genes Dev. 3:537-546), and the like.

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an anti sense orientation. That is, the DNA molecule isoperably linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to human phosphatidylserine synthase-like mRNA.Regulatory sequences operably linked to a nucleic acid cloned in theantisense orientation can be chosen to direct the continuous expressionof the antisense RNA molecule in a variety of cell types, for instanceviral promoters and/or enhancers, or regulatory sequences can be chosento direct constitutive, tissue-specific, or cell-type-specificexpression of antisense RNA. The antisense expression vector can be inthe form of a recombinant plasmid, phagemid, or attenuated virus inwhich antisense nucleic acids are produced under the control of a highefficiency regulatory region, the activity of which can be determined bythe cell type into which the vector is introduced. For a discussion ofthe regulation of gene expression using antisense genes see Weintraub etal. (1986) Reviews—Trends in Genetics, Vol. 1(1).

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2nd ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.) and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin, and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding a human phosphatidylserine synthase-like proteinor can be introduced on a separate vector. Cells stably transfected withthe introduced nucleic acid can be identified by drug selection (e.g.,cells that have incorporated the selectable marker gene will survive,while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) humanphosphatidylserine synthase-like protein. Accordingly, the inventionfurther provides methods for producing human phosphatidylserinesynthase-like protein using the host cells of the invention. In oneembodiment, the method comprises culturing the host cell of theinvention, into which a recombinant expression vector encoding a humanphosphatidylserine synthase-like protein has been introduced, in asuitable medium such that human phosphatidylserine synthase-like proteinis produced. In another embodiment, the method further comprisesisolating human phosphatidylserine synthase-like protein from the mediumor the host cell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichhuman phosphatidylserine synthase-like-coding sequences have beenintroduced. Such host cells can then be used to create nonhumantransgenic animals in which exogenous human phosphatidylserinesynthase-like sequences have been introduced into their genome orhomologous recombinant animals in which endogenous humanphosphatidylserine synthase-like sequences have been altered. Suchanimals are useful for studying the function and/or activity of humanphosphatidylserine synthase-like genes and proteins and for identifyingand/or evaluating modulators of human phosphatidylserine synthase-likeactivity. As used herein, a “transgenic animal” is a nonhuman animal,preferably a mammal, more preferably a rodent such as a rat or mouse, inwhich one or more of the cells of the animal includes a transgene. Otherexamples of transgenic animals include nonhuman primates, sheep, dogs,cows, goats, chickens, amphibians, etc. A transgene is exogenous DNAthat is integrated into the genome of a cell from which a transgenicanimal develops and which remains in the genome of the mature animal,thereby directing the expression of an encoded gene product in one ormore cell types or tissues of the transgenic animal. As used herein, a“homologous recombinant animal” is a nonhuman animal, preferably amammal, more preferably a mouse, in which an endogenous humanphosphatidylserine synthase-like gene has been altered by homologousrecombination between the endogenous gene and an exogenous DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal, prior to development of the animal.

A transgenic animal of the invention can be created by introducing humanphosphatidylserine synthase-like-encoding nucleic acid into the malepronuclei of a fertilized oocyte, e.g., by microinjection, retroviralinfection, and allowing the oocyte to develop in a pseudopregnant femalefoster animal. The human phosphatidylserine synthase-like cDNA sequencecan be introduced as a transgene into the genome of a nonhuman animal.Alternatively, a homologue of the mouse human phosphatidylserinesynthase-like gene can be isolated based on hybridization and used as atransgene. Intronic sequences and polyadenylation signals can also beincluded in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operablylinked to the human phosphatidylserine synthase-like transgene to directexpression of human phosphatidylserine synthase-like protein toparticular cells. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866, 4,870,009, and 4,873,191 and in Hogan (1986)Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1986). Similar methods are used for production ofother transgenic animals. A transgenic founder animal can be identifiedbased upon the presence of the human phosphatidylserine synthase-liketransgene in its genome and/or expression of human phosphatidylserinesynthase-like mRNA in tissues or cells of the animals. A transgenicfounder animal can then be used to breed additional animals carrying thetransgene. Moreover, transgenic animals carrying a transgene encodinghuman phosphatidylserine synthase-like gene can further be bred to othertransgenic animals carrying other transgenes.

To create a homologous recombinant animal, one prepares a vectorcontaining at least a portion of a human phosphatidylserinesynthase-like gene or a homolog of the gene into which a deletion,addition, or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the human phosphatidylserine synthase-like gene.In a preferred embodiment, the vector is designed such that, uponhomologous recombination, the endogenous human phosphatidylserinesynthase-like gene is functionally disrupted (i.e., no longer encodes afunctional protein; also referred to as a “knock out” vector).Alternatively, the vector can be designed such that, upon homologousrecombination, the endogenous human phosphatidylserine synthase-likegene is mutated or otherwise altered but still encodes functionalprotein (e.g., the upstream regulatory region can be altered to therebyalter the expression of the endogenous human phosphatidylserinesynthase-like protein). In the homologous recombination vector, thealtered portion of the human phosphatidylserine synthase-like gene isflanked at its 5′ and 3′ ends by additional nucleic acid of the humanphosphatidylserine synthase-like gene to allow for homologousrecombination to occur between the exogenous human phosphatidylserinesynthase-like gene carried by the vector and an endogenous humanphosphatidylserine synthase-like gene in an embryonic stem cell. Theadditional flanking human phosphatidylserine synthase-like nucleic acidis of sufficient length for successful homologous recombination with theendogenous gene. Typically, several kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the vector (see, e.g., Thomas andCapecchi (1987) Cell 51:503 for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation), and cells in which the introducedhuman phosphatidylserine synthase-like gene has homologously recombinedwith the endogenous human phosphatidylserine synthase-like gene areselected (see, e.g., Li et al. (1992) Cell 69:915). The selected cellsare then injected into a blastocyst of an animal (e.g., a mouse) to formaggregation chimeras (see, e.g., Bradley (1987) in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, ed. Robertson (IRL, Oxfordpp. 113-152). A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term.Progeny harboring the homologously recombined DNA in their germ cellscan be used to breed animals in which all cells of the animal containthe homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination vectors andhomologous recombinant animals are described further in Bradley (1991)Current Opinion in Bio/Techniques 2:823-829 and in PCT Publication Nos.WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169.

In another embodiment, transgenic nonhuman animals containing selectedsystems that allow for regulated expression of the transgene can beproduced. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355). If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the nonhuman transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and PCT Publication Nos. WO 97/07668 and WO 97/07669.

IV. Pharmaceutical Compositions

The human phosphatidylserine synthase-like nucleic acid molecules, humanphosphatidylserine synthase-like proteins, and anti-humanphosphatidylserine synthase-like antibodies (also referred to herein as“active compounds”) of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule, protein, orantibody and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

The compositions of the invention are useful to treat any of thedisorders discussed herein. Treatment is defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease or the predisposition toward disease.“Subject”, as used herein, can refer to a mammal, e.g. a human, or to anexperimental or animal or disease model. The subject can also be anon-human animal, e.g. a horse, cow, goat, or other domestic animal. Atherapeutic agent includes, but is not limited to, small molecules,peptides, antibodies, ribozymes and antisense oligonucleotides.

The compositions are provided in therapeutically effective amounts. By“therapeutically effective amounts” is intended an amount sufficient tomodulate the desired response. As defined herein, a therapeuticallyeffective amount of protein or polypeptide (i.e., an effective dosage)ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg bodyweight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of a protein, polypeptide, or antibody can include asingle treatment or, preferably, can include a series of treatments. Ina preferred example, a subject is treated with antibody, protein, orpolypeptide in the range of between about 0.1 to 20 mg/kg body weight,one time per week for between about 1 to 10 weeks, preferably between 2to 8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody, protein, or polypeptide used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays as described herein.

The present invention encompasses agents which modulate expression oractivity. An agent may, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e,. including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds.

It is understood that appropriate doses of small molecule agents dependsupon a number of factors within the skill of the ordinarily skilledphysician, veterinarian, or researcher. The dose(s) of the smallmolecule will vary, for example, depending upon the identity, size, andcondition of the subject or sample being treated, further depending uponthe route by which the composition is to be administered, if applicable,and the effect which the practitioner desires the small molecule to haveupon the nucleic acid or polypeptide of the invention. Exemplary dosesinclude milligram or microgram amounts of the small molecule perkilogram of subject or sample weight (e.g., about 1 microgram perkilogram to about 500 milligrams per kilogram, about 100 micrograms perkilogram to about 5 milligrams per kilogram, or about 1 microgram perkilogram to about 50 micrograms per kilogram. It is furthermoreunderstood that appropriate doses of a small molecule depend upon thepotency of the small molecule with respect to the expression or activityto be modulated. Such appropriate doses may be determined using theassays described herein. When one or more of these small molecules is tobe administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes, or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF; Parsippany, N.J.), or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion, and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a human phosphatidylserine synthase-like protein oranti-human phosphatidylserine synthase-like antibody) in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying, which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth, or gelatin; an excipientsuch as starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. For administrationby inhalation, the compounds are delivered in the form of an aerosolspray from a pressurized container or dispenser that contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art. Thecompounds can also be prepared in the form of suppositories (e.g., withconventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated with each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. Depending on thetype and severity of the disease, about 1 μg/kg to about 15 mg/kg (e.g.,0.1 to 20 mg/kg) of antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to about 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays. Anexemplary dosing regimen is disclosed in WO 94/04188. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470), or by stereotactic injection(see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods:(a) screening assays; (b) detection assays (e.g., chromosomal mapping,tissue typing, forensic biology); (c) predictive medicine (e.g.,diagnostic assays, prognostic assays, monitoring clinical trials, andpharmacogenomics); and (d) methods of treatment (e.g., therapeutic andprophylactic). The isolated nucleic acid molecules of the invention canbe used to express human phosphatidylserine synthase-like protein (e.g.,via a recombinant expression vector in a host cell in gene therapyapplications), to detect human phosphatidylserine synthase-like mRNA(e.g., in a biological sample) or a genetic lesion in a humanphosphatidylserine synthase-like gene, and to modulate humanphosphatidylserine synthase-like activity. In addition, the humanphosphatidylserine synthase-like proteins can be used to screen drugs orcompounds in disorders characterized by insufficient or excessiveproduction of human phosphatidylserine synthase-like protein orproduction of human phosphatidylserine synthase-like protein forms thathave decreased or aberrant activity compared to human phosphatidylserinesynthase-like wild type protein. In addition, the anti-humanphosphatidylserine synthase-like antibodies of the invention can be usedto detect and isolate human phosphatidylserine synthase-like proteinsand modulate human phosphatidylserine synthase-like activity.

A. Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules, or otherdrugs) that bind to human phosphatidylserine synthase-like proteins orhave a stimulatory or inhibitory effect on, for example, humanphosphatidylserine synthase-like expression or human phosphatidylserinesynthase-like activity.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including biological libraries, spatially addressable parallelsolid phase or solution phase libraries, synthetic library methodsrequiring deconvolution, the “one-bead one-compound” library method, andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, nonpeptide oligomer, orsmall molecule libraries of compounds (Lam (1997) Anticancer Drug Des.12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andGallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Bio/Techniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Pat.No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA89:1865-1869), or phage (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. USA 87:6378-6382; and Felici (1991) J. Mol. Biol.222:301-310).

Determining the ability of the test compound to bind to the humanphosphatidylserine synthase-like protein can be accomplished, forexample, by coupling the test compound with a radioisotope or enzymaticlabel such that binding of the test compound to the humanphosphatidylserine synthase-like protein or biologically active portionthereof can be determined by detecting the labeled compound in acomplex. For example, test compounds can be labeled with ¹²⁵I, ³⁵S, ¹⁴C,or ³H, either directly or indirectly, and the radioisotope detected bydirect counting of radioemmission or by scintillation counting.Alternatively, test compounds can be enzymatically labeled with, forexample, horseradish peroxidase, alkaline phosphatase, or luciferase,and the enzymatic label detected by determination of conversion of anappropriate substrate to product.

In a similar manner, one may determine the ability of the humanphosphatidylserine synthase-like protein to bind to or interact with ahuman phosphatidylserine synthase-like target molecule. By “targetmolecule” is intended a molecule with which a human phosphatidylserinesynthase-like protein binds or interacts in nature. In a preferredembodiment, the ability of the human phosphatidylserine synthase-likeprotein to bind to or interact with a human phosphatidylserinesynthase-like target molecule can be determined by monitoring theactivity of the target molecule. For example, the activity of the targetmolecule can be monitored by detecting catalytic/enzymatic activity ofthe target on an appropriate substrate, detecting the induction of areporter gene (e.g., a human phosphatidylserine synthase-like-responsiveregulatory element operably linked to a nucleic acid encoding adetectable marker, e.g. luciferase), or detecting a cellular response,for example, cellular differentiation or cell proliferation.

In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting a human phosphatidylserinesynthase-like protein or biologically active portion thereof with a testcompound and determining the ability of the test compound to bind to thehuman phosphatidylserine synthase-like protein or biologically activeportion thereof. Binding of the test compound to the humanphosphatidylserine synthase-like protein can be determined eitherdirectly or indirectly as described above. In a preferred embodiment,the assay includes contacting the human phosphatidylserine synthase-likeprotein or biologically active portion thereof with a known compoundthat binds human phosphatidylserine synthase-like protein to form anassay mixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to preferentially bind tohuman phosphatidylserine synthase-like protein or biologically activeportion thereof as compared to the known compound.

In another embodiment, an assay is a cell-free assay comprisingcontacting human phosphatidylserine synthase-like protein orbiologically active portion thereof with a test compound and determiningthe ability of the test compound to modulate (e.g., stimulate orinhibit) the activity of the human phosphatidylserine synthase-likeprotein or biologically active portion thereof. Determining the abilityof the test compound to modulate the activity of a humanphosphatidylserine synthase-like protein can be accomplished, forexample, by determining the ability of the human phosphatidylserinesynthase-like protein to bind to a human phosphatidylserinesynthase-like target molecule as described above for determining directbinding. In an alternative embodiment, determining the ability of thetest compound to modulate the activity of a human phosphatidylserinesynthase-like protein can be accomplished by determining the ability ofthe human phosphatidylserine synthase-like protein to further modulate ahuman phosphatidylserine synthase-like target molecule. For example, thecatalytic/enzymatic activity of the target molecule on an appropriatesubstrate can be determined as previously described.

In yet another embodiment, the cell-free assay comprises contacting thehuman phosphatidylserine synthase-like protein or biologically activeportion thereof with a known compound that binds a humanphosphatidylserine synthase-like protein to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to preferentially bind to or modulate theactivity of a human phosphatidylserine synthase-like target molecule.

In the above-mentioned assays, it may be desirable to immobilize eithera human phosphatidylserine synthase-like protein or its target moleculeto facilitate separation of complexed from uncomplexed forms of one orboth of the proteins, as well as to accommodate automation of the assay.In one embodiment, a fusion protein can be provided that adds a domainthat allows one or both of the proteins to be bound to a matrix. Forexample, glutathione-S-transferase/human phosphatidylserinesynthase-like fusion proteins or glutathione-S-transferase/target fusionproteins can be adsorbed onto glutathione sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione-derivatized microtitre plates,which are then combined with the test compound or the test compound andeither the nonadsorbed target protein or human phosphatidylserinesynthase-like protein, and the mixture incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads or microtitre plate wellsare washed to remove any unbound components and complex formation ismeasured either directly or indirectly, for example, as described above.Alternatively, the complexes can be dissociated from the matrix, and thelevel of human phosphatidylserine synthase-like binding or activitydetermined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either humanphosphatidylserine synthase-like protein or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin.Biotinylated human phosphatidylserine synthase-like molecules or targetmolecules can be prepared from biotin-NHS (N-hydroxy-succinimide) usingtechniques well known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96-well plates (Pierce Chemicals). Alternatively,antibodies reactive with a human phosphatidylserine synthase-likeprotein or target molecules but which do not interfere with binding ofthe human phosphatidylserine synthase-like protein to its targetmolecule can be derivatized to the wells of the plate, and unboundtarget or human phosphatidylserine synthase-like protein trapped in thewells by antibody conjugation. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with thehuman phosphatidylserine synthase-like protein or target molecule, aswell as enzyme-linked assays that rely on detecting an enzymaticactivity associated with the human phosphatidylserine synthase-likeprotein or target molecule.

In another embodiment, modulators of human phosphatidylserinesynthase-like expression are identified in a method in which a cell iscontacted with a candidate compound and the expression of humanphosphatidylserine synthase-like mRNA or protein in the cell isdetermined relative to expression of human phosphatidylserinesynthase-like mRNA or protein in a cell in the absence of the candidatecompound. When expression is greater (statistically significantlygreater) in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of humanphosphatidylserine synthase-like mRNA or protein expression.Alternatively, when expression is less (statistically significantlyless) in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of humanphosphatidylserine synthase-like mRNA or protein expression. The levelof human phosphatidylserine synthase-like mRNA or protein expression inthe cells can be determined by methods described herein for detectinghuman phosphatidylserine synthase-like mRNA or protein.

In yet another aspect of the invention, the human phosphatidylserinesynthase-like proteins can be used as “bait proteins” in a two-hybridassay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervoset al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.268:12046-12054; Bartel et al. (1993) Bio/Techniques 14:920-924;Iwabuchi et al. (1993) Oncogene 8:1693-1696; and PCT Publication No. WO94/10300), to identify other proteins, which bind to or interact withhuman phosphatidylserine synthase-like protein (“humanphosphatidylserine synthase-like-binding proteins” or “humanphosphatidylserine synthase-like-bp”) and modulate humanphosphatidylserine synthase-like activity. Such human phosphatidylserinesynthase-like-binding proteins are also likely to be involved in thepropagation of signals by the human phosphatidylserine synthase-likeproteins as, for example, upstream or downstream elements of the humanphosphatidylserine synthase-like pathway.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(1) map their respective genes on a chromosome; (2) identify anindividual from a minute biological sample (tissue typing); and (3) aidin forensic identification of a biological sample. These applicationsare described in the subsections below.

1. Chromosome Mapping

The isolated complete or partial human phosphatidylserine synthase-likegene sequences of the invention can be used to map their respectivehuman phosphatidylserine synthase-like genes on a chromosome, therebyfacilitating the location of gene regions associated with geneticdisease. Computer analysis of human phosphatidylserine synthase-likesequences can be used to rapidly select PCR primers (preferably 15-25 bpin length) that do not span more than one exon in the genomic DNA,thereby simplifying the amplification process. These primers can then beused for PCR screening of somatic cell hybrids containing individualhuman chromosomes. Only those hybrids containing the human genecorresponding to the human phosphatidylserine synthase-like sequenceswill yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow (because they lack a particular enzyme), but in whichhuman cells can, the one human chromosome that contains the geneencoding the needed enzyme will be retained. By using various media,panels of hybrid cell lines can be established. Each cell line in apanel contains either a single human chromosome or a small number ofhuman chromosomes, and a full set of mouse chromosomes, allowing easymapping of individual genes to specific human chromosomes (D'Eustachioet al. (1983) Science 220:919-924). Somatic cell hybrids containing onlyfragments of human chromosomes can also be produced by using humanchromosomes with translocations and deletions.

Other mapping strategies that can similarly be used to map a humanphosphatidylserine synthase-like sequence to its chromosome include insitu hybridization (described in Fan et al. (1990) Proc. Natl. Acad.Sci. USA 87:6223-27), pre-screening with labeled flow-sortedchromosomes, and pre-selection by hybridization to chromosome specificcDNA libraries. Furthermore, fluorescence in situ hybridization (FISH)of a DNA sequence to a metaphase chromosomal spread can be used toprovide a precise chromosomal location in one step. For a review of thistechnique, see Verma et al. (1988) Human Chromosomes: A Manual of BasicTechniques (Pergamon Press, NY). The FISH technique can be used with aDNA sequence as short as 500 or 600 bases. However, clones larger than1,000 bases have a higher likelihood of binding to a unique chromosomallocation with sufficient signal intensity for simple detection.Preferably 1,000 bases, and more preferably 2,000 bases will suffice toget good results in a reasonable amount of time.

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Another strategy to map the chromosomal location of humanphosphatidylserine synthase-like genes uses human phosphatidylserinesynthase-like polypeptides and fragments and sequences of the presentinvention and antibodies specific thereto. This mapping can be carriedout by specifically detecting the presence of a human phosphatidylserinesynthase-like polypeptide in members of a panel of somatic cell hybridsbetween cells of a first species of animal from which the proteinoriginates and cells from a second species of animal, and thendetermining which somatic cell hybrid(s) expresses the polypeptide andnoting the chromosomes(s) from the first species of animal that itcontains. For examples of this technique, see Pajunen et al. (1988)Cytogenet. Cell. Genet. 47:37-41 and Van Keuren et al. (1986) Hum.Genet. 74:34-40. Alternatively, the presence of a humanphosphatidylserine synthase-like polypeptide in the somatic cell hybridscan be determined by assaying an activity or property of thepolypeptide, for example, enzymatic activity, as described inBordelon-Riser et al. (1979) Somatic Cell Genetics 5:597-613 andOwerbach et al. (1978) Proc. Natl. Acad. Sci. USA 75:5640-5644.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship betweengenes and disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, e.g., Egeland et al. (1987) Nature325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the humanphosphatidylserine synthase-like gene can be determined. If a mutationis observed in some or all of the affected individuals but not in anyunaffected individuals, then the mutation is likely to be the causativeagent of the particular disease. Comparison of affected and unaffectedindividuals generally involves first looking for structural alterationsin the chromosomes such as deletions or translocations that are visiblefrom chromosome spreads or detectable using PCR based on that DNAsequence. Ultimately, complete sequencing of genes from severalindividuals can be performed to confirm the presence of a mutation andto distinguish mutations from polymorphisms.

2. Tissue Typing

The human phosphatidylserine synthase-like sequences of the presentinvention can also be used to identify individuals from minutebiological samples. The United States military, for example, isconsidering the use of restriction fragment length polymorphism (RFLP)for identification of its personnel. In this technique, an individual'sgenomic DNA is digested with one or more restriction enzymes and probedon a Southern blot to yield unique bands for identification. Thesequences of the present invention are useful as additional DNA markersfor RFLP (described in U.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique for determining the actual base-by-baseDNA sequence of selected portions of an individual's genome. Thus, thehuman phosphatidylserine synthase-like sequences of the invention can beused to prepare two PCR primers from the 5′ and 3′ ends of thesequences. These primers can then be used to amplify an individual's DNAand subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The human phosphatidylserine synthase-like sequences of theinvention uniquely represent portions of the human genome. Allelicvariation occurs to some degree in the coding regions of thesesequences, and to a greater degree in the noncoding regions. It isestimated that allelic variation between individual humans occurs with afrequency of about once per each 500 bases. Each of the sequencesdescribed herein can, to some degree, be used as a standard againstwhich DNA from an individual can be compared for identificationpurposes. The noncoding sequences of SEQ ID NO:5 can comfortably providepositive individual identification with a panel of perhaps 10 to 1,000primers that each yield a noncoding amplified sequence of 100 bases. Ifa predicted coding sequence, such as that in SEQ ID NO:5, is used, amore appropriate number of primers for positive individualidentification would be 500 to 2,000.

3. Use of Partial Human Phosphatidylserine Synthase-Like Sequences inForensic Biology

DNA-based identification techniques can also be used in forensicbiology. In this manner, PCR technology can be used to amplify DNAsequences taken from very small biological samples such as tissues,e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen foundat a crime scene. The amplified sequence can then be compared to astandard, thereby allowing identification of the origin of thebiological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” that is unique to a particular individual. Asmentioned above, actual base sequence information can be used foridentification as an accurate alternative to patterns formed byrestriction enzyme generated fragments. Sequences targeted to noncodingregions of SEQ ID NO:5 are particularly appropriate for this use asgreater numbers of polymorphisms occur in the noncoding regions, makingit easier to differentiate individuals using this technique. Examples ofpolynucleotide reagents include the human phosphatidylserinesynthase-like sequences or portions thereof, e.g., fragments derivedfrom the noncoding regions of SEQ ID NO:5 having a length of at least 20or 30 bases.

The human phosphatidylserine synthase-like sequences described hereincan further be used to provide polynucleotide reagents, e.g., labeled orlabelable probes that can be used in, for example, an in situhybridization technique, to identify a specific tissue. This can be veryuseful in cases where a forensic pathologist is presented with a tissueof unknown origin. Panels of such human phosphatidylserine synthase-likeprobes, can be used to identify tissue by species and/or by organ type.

In a similar fashion, these reagents, e.g., human phosphatidylserinesynthase-like primers or probes can be used to screen tissue culture forcontamination (i.e., screen for the presence of a mixture of differenttypes of cells in a culture).

C. Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trails are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. These applications aredescribed in the subsections below.

1. Diagnostic Assays

One aspect of the present invention relates to diagnostic assays fordetecting human phosphatidylserine synthase-like protein and/or nucleicacid expression as well as human phosphatidylserine synthase-likeactivity, in the context of a biological sample. An exemplary method fordetecting the presence or absence of human phosphatidylserinesynthase-like proteins in a biological sample involves obtaining abiological sample from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting humanphosphatidylserine synthase-like protein or nucleic acid (e.g., mRNA,genomic DNA) that encodes human phosphatidylserine synthase-like proteinsuch that the presence of human phosphatidylserine synthase-like proteinis detected in the biological sample. Results obtained with a biologicalsample from the test subject may be compared to results obtained with abiological sample from a control subject.

A preferred agent for detecting human phosphatidylserine synthase-likemRNA or genomic DNA is a labeled nucleic acid probe capable ofhybridizing to human phosphatidylserine synthase-like mRNA or genomicDNA. The nucleic acid probe can be, for example, a full-length humanphosphatidylserine synthase-like nucleic acid, such as the nucleic acidof SEQ ID NO:5, or a portion thereof, such as a nucleic acid molecule ofat least 15, 30, 50, 100, 250, or 500 nucleotides in length andsufficient to specifically hybridize under stringent conditions to humanphosphatidylserine synthase-like mRNA or genomic DNA. Other suitableprobes for use in the diagnostic assays of the invention are describedherein.

A preferred agent for detecting human phosphatidylserine synthase-likeprotein is an antibody capable of binding to human phosphatidylserinesynthase-like protein, preferably an antibody with a detectable label.Antibodies can be polyclonal, or more preferably, monoclonal. An intactantibody, or a fragment thereof (e.g., Fab or F(ab)₂) can be used. Theterm “labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin.

The term “biological sample” is intended to include tissues, cells, andbiological fluids isolated from a subject, as well as tissues, cells,and fluids present within a subject. That is, the detection method ofthe invention can be used to detect human phosphatidylserinesynthase-like mRNA, protein, or genomic DNA in a biological sample invitro as well as in vivo. For example, in vitro techniques for detectionof human phosphatidylserine synthase-like mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetection of human phosphatidylserine synthase-like protein includeenzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, and immunofluorescence. In vitro techniques fordetection of human phosphatidylserine synthase-like genomic DNA includeSouthern hybridizations. Furthermore, in vivo techniques for detectionof human phosphatidylserine synthase-like protein include introducinginto a subject a labeled anti-human phosphatidylserine synthase-likeantibody. For example, the antibody can be labeled with a radioactivemarker whose presence and location in a subject can be detected bystandard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a peripheral blood samplecontaining erythrocytes, lymphocytes and platelets which make for easilyassayable cells.

The invention also encompasses kits for detecting the presence of humanphosphatidylserine synthase-like proteins in a biological sample (a testsample). Such kits can be used to determine if a subject is sufferingfrom or is at increased risk of developing a disorder associated withaberrant expression of human phosphatidylserine synthase-like protein(e.g., an immunological disorder). For example, the kit can comprise alabeled compound or agent capable of detecting human phosphatidylserinesynthase-like protein or mRNA in a biological sample and means fordetermining the amount of a human phosphatidylserine synthase-likeprotein in the sample (e.g., an anti-human phosphatidylserinesynthase-like antibody or an oligonucleotide probe that binds to DNAencoding a human phosphatidylserine synthase-like protein, e.g., SEQ IDNO:6). Kits can also include instructions for observing that the testedsubject is suffering from or is at risk of developing a disorderassociated with aberrant expression of human phosphatidylserinesynthase-like sequences if the amount of human phosphatidylserinesynthase-like protein or mRNA is above or below a normal level.

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) that binds to humanphosphatidylserine synthase-like protein; and, optionally, (2) a second,different antibody that binds to human phosphatidylserine synthase-likeprotein or the first antibody and is conjugated to a detectable agent.For oligonucleotide-based kits, the kit can comprise, for example: (1)an oligonucleotide, e.g., a detectably labeled oligonucleotide, thathybridizes to a human phosphatidylserine synthase-like nucleic acidsequence or (2) a pair of primers useful for amplifying a humanphosphatidylserine synthase-like nucleic acid molecule.

The kit can also comprise, e.g., a buffering agent, a preservative, or aprotein stabilizing agent. The kit can also comprise componentsnecessary for detecting the detectable agent (e.g., an enzyme or asubstrate). The kit can also contain a control sample or a series ofcontrol samples that can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container, and all of the various containers are within asingle package along with instructions for observing whether the testedsubject is suffering from or is at risk of developing a disorderassociated with aberrant expression of human phosphatidylserinesynthase-like proteins.

2. Other Diagnostic Assays

In another aspect, the invention features a method of analyzing aplurality of capture probes. The method can be used, e.g., to analyzegene expression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g., anucleic acid or peptide sequence; contacting the array with aphosphatidylserine synthase-like nucleic acid, preferably purified,polypeptide, preferably purified, or antibody, and thereby evaluatingthe plurality of capture probes. Binding, e.g., in the case of a nucleicacid, hybridization, with a capture probe at an address of theplurality, is detected, e.g., by signal generated from a label attachedto the phosphatidylserine synthase-like nucleic acid, polypeptide, orantibody. The capture probes can be a set of nucleic acids from aselected sample, e.g., a sample of nucleic acids derived from a controlor non-stimulated tissue or cell.

The method can include contacting the phosphatidylserine synthase-likenucleic acid, polypeptide, or antibody with a first array having aplurality of capture probes and a second array having a differentplurality of capture probes. The results of each hybridization can becompared, e.g., to analyze differences in expression between a first andsecond sample. The first plurality of capture probes can be from acontrol sample, e.g., a wild type, normal, or non-diseased,non-stimulated, sample, e.g., a biological fluid, tissue, or cellsample. The second plurality of capture probes can be from anexperimental sample, e.g., a mutant type, at risk, disease-state ordisorder-state, or stimulated, sample, e.g., a biological fluid, tissue,or cell sample.

The plurality of capture probes can be a plurality of nucleic acidprobes each of which specifically hybridizes, with an allele of aphosphatidylserine synthase-like sequence of the invention. Such methodscan be used to diagnose a subject, e.g., to evaluate risk for a diseaseor disorder, to evaluate suitability of a selected treatment for asubject, to evaluate whether a subject has a disease or disorder. Thus,for example, the 32670 sequence set forth in SEQ ID NO:5 and SEQ ID NO:7encodes a phosphatidylserine synthase-like polypeptide that is useful toevaluate a disease or disorder wherein there is defective cell membraneformation.

The method can be used to detect single nucleotide polymorphisms (SNPs),as described below.

In another aspect, the invention features a method of analyzing aplurality of probes. The method is useful, e.g., for analyzing geneexpression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the pluralityhaving a unique capture probe, e.g., wherein the capture probes are froma cell or subject which express a phosphatidylserine synthase-likepolypeptide of the invention or from a cell or subject in which aphosphatidylserine synthase-like-mediated response has been elicited,e.g., by contact of the cell with a phosphatidylserine synthase-likenucleic acid or protein of the invention, or administration to the cellor subject a phosphatidylserine synthase-like nucleic acid or protein ofthe invention; contacting the array with one or more inquiry probes,wherein an inquiry probe can be a nucleic acid, polypeptide, or antibody(which is preferably other than a phosphatidylserine synthase-likenucleic acid, polypeptide, or antibody of the invention); providing atwo dimensional array having a plurality of addresses, each address ofthe plurality being positionally distinguishable from each other addressof the plurality, and each address of the plurality having a uniquecapture probe, e.g., wherein the capture probes are from a cell orsubject which does not express a phosphatidylserine synthase-likesequence of the invention (or does not express as highly as in the caseof the phosphatidylserine synthase-like positive plurality of captureprobes) or from a cell or subject in which a phosphatidylserinesynthase-like-mediated response has not been elicited (or has beenelicited to a lesser extent than in the first sample); contacting thearray with one or more inquiry probes (which is preferably other than aphosphatidylserine synthase-like nucleic acid, polypeptide, or antibodyof the invention), and thereby evaluating the plurality of captureprobes. Binding, e.g., in the case of a nucleic acid, hybridization,with a capture probe at an address of the plurality, is detected, e.g.,by signal generated from a label attached to the nucleic acid,polypeptide, or antibody.

In another aspect, the invention features a method of analyzing aphosphatidylserine synthase-like sequence of the invention, e.g.,analyzing structure, function, or relatedness to other nucleic acid oramino acid sequences. The method includes: providing aphosphatidylserine synthase-like nucleic acid or amino acid sequence,e.g., the 32670 sequence set forth in SEQ ID NO:5, SEQ ID NO:7, or aportion thereof; comparing the phosphatidylserine synthase-like sequencewith one or more preferably a plurality of sequences from a collectionof sequences, e.g., a nucleic acid or protein sequence database; tothereby analyze the phosphatidylserine synthase-like sequence of theinvention.

The method can include evaluating the sequence identity between aphosphatidylserine synthase-like sequence of the invention, e.g., the32670 sequence, and a database sequence. The method can be performed byaccessing the database at a second site, e.g., over the internet.

In another aspect, the invention features, a set of oligonucleotides,useful, e.g., for identifying SNP's, or identifying specific alleles ofa Phosphatidylserine synthase-like sequence of the invention, e.g., the15571 sequence. The set includes a plurality of oligonucleotides, eachof which has a different nucleotide at an interrogation position, e.g.,an SNP or the site of a mutation. In a preferred embodiment, theoligonucleotides of the plurality identical in sequence with one another(except for differences in length). The oligonucleotides can be providedwith differential labels, such that an oligonucleotides which hybridizesto one allele provides a signal that is distinguishable from anoligonucleotides which hybridizes to a second allele.

3. Prognostic Assays

The methods described herein can furthermore be utilized as diagnosticor prognostic assays to identify subjects having or at risk ofdeveloping a disease or disorder associated with humanphosphatidylserine synthase-like protein, human phosphatidylserinesynthase-like nucleic acid expression, or human phosphatidylserinesynthase-like activity. Prognostic assays can be used for prognostic orpredictive purposes to thereby prophylactically treat an individualprior to the onset of a disorder characterized by or associated withhuman phosphatidylserine synthase-like protein, human phosphatidylserinesynthase-like nucleic acid expression, or human phosphatidylserinesynthase-like activity.

Thus, the present invention provides a method in which a test sample isobtained from a subject, and human phosphatidylserine synthase-likeprotein or nucleic acid (e.g., mRNA, genomic DNA) is detected, whereinthe presence of human phosphatidylserine synthase-like protein ornucleic acid is diagnostic for a subject having or at risk of developinga disease or disorder associated with aberrant human phosphatidylserinesynthase-like expression or activity. As used herein, a “test sample”refers to a biological sample obtained from a subject of interest. Forexample, a test sample can be a biological fluid (e.g., serum), cellsample, or tissue.

Furthermore, using the prognostic assays described herein, the presentinvention provides methods for determining whether a subject can beadministered a specific agent (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, nucleic acid, small molecule, or otherdrug candidate) or class of agents (e.g., agents of a type that decreasehuman phosphatidylserine synthase-like activity) to effectively treat adisease or disorder associated with aberrant human phosphatidylserinesynthase-like expression or activity. In this manner, a test sample isobtained and human phosphatidylserine synthase-like protein or nucleicacid is detected. The presence of human phosphatidylserine synthase-likeprotein or nucleic acid is diagnostic for a subject that can beadministered the agent to treat a disorder associated with aberranthuman phosphatidylserine synthase-like expression or activity.

The methods of the invention can also be used to detect genetic lesionsor mutations in a human phosphatidylserine synthase-like gene. Inpreferred embodiments, the methods include detecting, in a sample ofcells from the subject, the presence or absence of a genetic lesion ormutation characterized by at least one of an alteration affecting theintegrity of a gene encoding a human phosphatidylserinesynthase-like-protein, or the misexpression of the humanphosphatidylserine synthase-like gene. For example, such genetic lesionsor mutations can be detected by ascertaining the existence of at leastone of: (1) a deletion of one or more nucleotides from a humanphosphatidylserine synthase-like gene; (2) an addition of one or morenucleotides to a human phosphatidylserine synthase-like gene; (3) asubstitution of one or more nucleotides of a human phosphatidylserinesynthase-like gene; (4) a chromosomal rearrangement of a humanphosphatidylserine synthase-like gene; (5) an alteration in the level ofa messenger RNA transcript of a human phosphatidylserine synthase-likegene; (6) an aberrant modification of a human phosphatidylserinesynthase-like gene, such as of the methylation pattern of the genomicDNA; (7) the presence of a non-wild-type splicing pattern of a messengerRNA transcript of a human phosphatidylserine synthase-like gene; (8) anon-wild-type level of a human phosphatidylserine synthase-like-protein;(9) an allelic loss of a human phosphatidylserine synthase-like gene;and (10) an inappropriate post-translational modification of a humanphosphatidylserine synthase-like-protein. As described herein, there area large number of assay techniques known in the art that can be used fordetecting lesions in a human phosphatidylserine synthase-like gene. Anycell type or tissue, preferably peripheral blood leukocytes, in whichhuman phosphatidylserine synthase-like proteins are expressed may beutilized in the prognostic assays described herein.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the humanphosphatidylserine synthase-like-gene (see, e.g., Abravaya et al. (1995)Nucleic Acids Res. 23:675-682). It is anticipated that PCR and/or LCRmay be desirable to use as a preliminary amplification step inconjunction with any of the techniques used for detecting mutationsdescribed herein.

Alternative amplification methods include self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a human phosphatidylserinesynthase-like gene from a sample cell can be identified by alterationsin restriction enzyme cleavage patterns of isolated test sample andcontrol DNA digested with one or more restriction endonucleases.Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Pat.No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in a human phosphatidylserinesynthase-like molecule can be identified by hybridizing a sample andcontrol nucleic acids, e.g., DNA or RNA, to high density arrayscontaining hundreds or thousands of oligonucleotides probes (Cronin etal. (1996) Human Mutation 7:244-255; Kozal et al. (1996) Nature Medicine2:753-759). In yet another embodiment, any of a variety of sequencingreactions known in the art can be used to directly sequence the humanphosphatidylserine synthase-like gene and detect mutations by comparingthe sequence of the sample human phosphatidylserine synthase-like genewith the corresponding wild-type (control) sequence. Examples ofsequencing reactions include those based on techniques developed byMaxim and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplatedthat any of a variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays ((1995) Bio/Techniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT PublicationNo. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; andGriffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

Other methods for detecting mutations in the human phosphatidylserinesynthase-like gene include methods in which protection from cleavageagents is used to detect mismatched bases in RNA/RNA or RNA/DNAheteroduplexes (Myers et al. (1985) Science 230:1242). See, also Cottonet al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992)Methods Enzymol. 217:286-295. In a preferred embodiment, the control DNAor RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more “DNA mismatch repair” enzymes that recognize mismatched basepairs in double-stranded DNA in defined systems for detecting andmapping point mutations in human phosphatidylserine synthase-like cDNAsobtained from samples of cells. See, e.g., Hsu et al. (1994)Carcinogenesis 15:1657-1662. According to an exemplary embodiment, aprobe based on a human phosphatidylserine synthase-like sequence, e.g.,a wild-type human phosphatidylserine synthase-like sequence, ishybridized to a cDNA or other DNA product from a test cell(s). Theduplex is treated with a DNA mismatch repair enzyme, and the cleavageproducts, if any, can be detected from electrophoresis protocols or thelike. See, e.g., U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in human phosphatidylserine synthase-likegenes. For example, single-strand conformation polymorphism (SSCP) maybe used to detect differences in electrophoretic mobility between mutantand wild-type nucleic acids (Orita et al. (1989) Proc. Natl. Acad. Sci.USA 86:2766; see also Cotton (1993) Mutat. Res. 285:125-144; Hayashi(1992) Genet. Anal. Tech. Appl. 9:73-79). The sensitivity of the assaymay be enhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double-stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditions thatpermit hybridization only if a perfect match is found (Saiki et al.(1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci. USA86:6230). Such allele-specific oligonucleotides are hybridized toPCR-amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele-specific amplification technology, which dependson selective PCR amplification, may be used in conjunction with theinstant invention. Oligonucleotides used as primers for specificamplification may carry the mutation of interest in the center of themolecule so that amplification depends on differential hybridization(Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme3′ end of one primer where, under appropriate conditions, mismatch canprevent or reduce polymerase extension (Prossner (1993) Tibtech 11:238).In addition, it may be desirable to introduce a novel restriction sitein the region of the mutation to create cleavage-based detection(Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated thatin certain embodiments amplification may also be performed using Taqligase for amplification (Barany (1991) Proc. Natl. Acad. Sci. USA88:189). In such cases, ligation will occur only if there is a perfectmatch at the 3′ end of the 5′ sequence making it possible to detect thepresence of a known mutation at a specific site by looking for thepresence or absence of amplification.

The methods described herein may be performed, for example, by utilizingprepackaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnosed patients exhibiting symptoms orfamily history of a disease or illness involving a humanphosphatidylserine synthase-like gene.

4. Pharmacogenomics

Agents, or modulators that have a stimulatory or inhibitory effect onhuman phosphatidylserine synthase-like activity (e.g., humanphosphatidylserine synthase-like gene expression) as identified by ascreening assay described herein, can be administered to individuals totreat (prophylactically or therapeutically) disorders associated withaberrant human phosphatidylserine synthase-like activity. In conjunctionwith such treatment, the pharmacogenomics (i.e., the study of therelationship between an individual's genotype and that individual'sresponse to a foreign compound or drug) of the individual may beconsidered. Differences in metabolism of therapeutics can lead to severetoxicity or therapeutic failure by altering the relation between doseand blood concentration of the pharmacologically active drug. Thus, thepharmacogenomics of the individual permits the selection of effectiveagents (e.g., drugs) for prophylactic or therapeutic treatments based ona consideration of the individual's genotype. Such pharmacogenomics canfurther be used to determine appropriate dosages and therapeuticregimens. Accordingly, the activity of human phosphatidylserinesynthase-like protein, expression of human phosphatidylserinesynthase-like nucleic acid, or mutation content of humanphosphatidylserine synthase-like genes in an individual can bedetermined to thereby select appropriate agent(s) for therapeutic orprophylactic treatment of the individual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Linder (1997) Clin. Chem.43(2):254-266. In general, two types of pharmacogenetic conditions canbe differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body are referred to as “altered drugaction.” Genetic conditions transmitted as single factors altering theway the body acts on drugs are referred to as “altered drug metabolism”.These pharmacogenetic conditions can occur either as rare defects or aspolymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency(G6PD) is a common inherited enzymopathy in which the main clinicalcomplication is haemolysis after ingestion of oxidant drugs(antimalarials, sulfonamides, analgesics, nitrofurans) and consumptionof fava beans.

Differences in metabolism of therapeutics can lead to severe toxicity ortherapeutic failure by altering the relation between dose and bloodconcentration of the pharmacologically active drug. Thus, a physician orclinician may consider applying knowledge obtained in relevantpharmacogenomics studies in determining whether to administer aphosphatidylserine synthase-like molecule or phosphatidylserinesynthase-like modulator of the invention as well as tailoring the dosageand/or therapeutic regimen of treatment with a phosphatidylserinesynthase-like molecule or phosphatidylserine synthase-like modulator ofthe invention.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, an “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drug's target is known (e.g., aphosphatidylserine synthase-like protein of the present invention), allcommon variants of that gene can be fairly easily identified in thepopulation and it can be determined if having one version of the geneversus another is associated with a particular drug response.

Alternatively, a method termed the “gene expression profiling”, can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., aphosphatidylserine synthase-like molecule or phosphatidylserinesynthase-like modulator of the present invention) can give an indicationwhether gene pathways related to toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment of an individual.This knowledge, when applied to dosing or drug selection, can avoidadverse reactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with aphosphatidylserine synthase-like molecule or phosphatidylserinesynthase-like modulator of the invention, such as a modulator identifiedby one of the exemplary screening assays described herein.

The present invention further provides methods for identifying newagents, or combinations, that are based on identifying agents thatmodulate the activity of one or more of the gene products encoded by oneor more of the Phosphatidylserine synthase-like genes of the presentinvention, wherein these products may be associated with resistance ofthe cells to a therapeutic agent. Specifically, the activity of theproteins encoded by the phosphatidylserine synthase-like genes of thepresent invention can be used as a basis for identifying agents forovercoming agent resistance. By blocking the activity of one or more ofthe resistance proteins, target cells, e.g., hepatic stellate cells,will become sensitive to treatment with an agent that the unmodifiedtarget cells were resistant to.

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a phosphatidylserine synthase-like protein can be applied inclinical trials. For example, the effectiveness of an agent determinedby a screening assay as described herein to increase phosphatidylserinesynthase-like gene expression, protein levels, or upregulatephosphatidylserine synthase-like activity, can be monitored in clinicaltrials of subjects exhibiting decreased phosphatidylserine synthase-likegene expression, protein levels, or downregulated phosphatidylserinesynthase-like activity. Alternatively, the effectiveness of an agentdetermined by a screening assay to decrease phosphatidylserinesynthase-like gene expression, protein levels, or downregulatephosphatidylserine synthase-like activity, can be monitored in clinicaltrials of subjects exhibiting increased phosphatidylserine synthase-likegene expression, protein levels, or upregulated phosphatidylserinesynthase-like activity. In such clinical trials, the expression oractivity of a phosphatidylserine synthase-like gene, and preferably,other genes that have been implicated in, for example, aphosphatidylserine synthase-like-associated disorder can be used as a“read out” or markers of the phenotype of a particular cell.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, a PM will show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the activity of human phosphatidylserine synthase-like protein,expression of human phosphatidylserine synthase-like nucleic acid, ormutation content of human phosphatidylserine synthase-like genes in anindividual can be determined to thereby select appropriate agent(s) fortherapeutic or prophylactic treatment of the individual. In addition,pharmacogenetic studies can be used to apply genotyping of polymorphicalleles encoding drug-metabolizing enzymes to the identification of anindividual's drug responsiveness phenotype. This knowledge, when appliedto dosing or drug selection, can avoid adverse reactions or therapeuticfailure and thus enhance therapeutic or prophylactic efficiency whentreating a subject with a human phosphatidylserine synthase-likemodulator, such as a modulator identified by one of the exemplaryscreening assays described herein.

5. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of human phosphatidylserine synthase-like genescan be applied not only in basic drug screening but also in clinicaltrials. For example, the effectiveness of an agent, as determined by ascreening assay as described herein, to increase or decrease humanphosphatidylserine synthase-like gene expression, protein levels, orprotein activity, can be monitored in clinical trials of subjectsexhibiting decreased or increased human phosphatidylserine synthase-likegene expression, protein levels, or protein activity.

For example, and not by way of limitation, genes that are modulated incells by treatment with an agent (e.g., compound, drug, or smallmolecule) that modulates human phosphatidylserine synthase-like activity(e.g., as identified in a screening assay described herein) can beidentified. Thus, to study the effect of agents on cellularproliferation disorders, for example, in a clinical trial, cells can beisolated and RNA prepared and analyzed for the levels of expression ofhuman phosphatidylserine synthase-like genes and other genes implicatedin the disorder. The levels of gene expression (i.e., a gene expressionpattern) can be quantified by Northern blot analysis or RT-PCR, asdescribed herein, or alternatively by measuring the amount of proteinproduced, by one of the methods as described herein, or by measuring thelevels of activity of human phosphatidylserine synthase-like genes orother genes. In this way, the gene expression pattern can serve as amarker, indicative of the physiological response of the cells to theagent. Accordingly, this response state may be determined before, and atvarious points during, treatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (1) obtaininga preadministration sample from a subject prior to administration of theagent; (2) detecting the level of expression of a humanphosphatidylserine synthase-like protein, mRNA, or genomic DNA in thepreadministration sample; (3) obtaining one or more postadministrationsamples from the subject; (4) detecting the level of expression oractivity of the human phosphatidylserine synthase-like protein, mRNA, orgenomic DNA in the postadministration samples; (5) comparing the levelof expression or activity of the human phosphatidylserine synthase-likeprotein, mRNA, or genomic DNA in the preadministration sample with thehuman phosphatidylserine synthase-like protein, mRNA, or genomic DNA inthe postadministration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly to bring aboutthe desired effect, i.e., for example, an increase or a decrease in theexpression or activity of a human phosphatidylserine synthase-likeprotein.

C. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant human phosphatidylserinesynthase-like expression or activity. Additionally, the compositions ofthe invention find use in the treatment of disorders described herein.Thus, therapies for disorders associated with CCC are encompassedherein.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject a disease or condition associated with an aberrant humanphosphatidylserine synthase-like expression or activity by administeringto the subject an agent that modulates human phosphatidylserinesynthase-like expression or at least one human phosphatidylserinesynthase-like gene activity. Subjects at risk for a disease that iscaused, or contributed to, by aberrant human phosphatidylserinesynthase-like expression or activity can be identified by, for example,any or a combination of diagnostic or prognostic assays as describedherein. Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the human phosphatidylserinesynthase-like aberrancy, such that a disease or disorder is preventedor, alternatively, delayed in its progression. Depending on the type ofhuman phosphatidylserine synthase-like aberrancy, for example, a humanphosphatidylserine synthase-like agonist or human phosphatidylserinesynthase-like antagonist agent can be used for treating the subject. Theappropriate agent can be determined based on screening assays describedherein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating humanphosphatidylserine synthase-like expression or activity for therapeuticpurposes. The modulatory method of the invention involves contacting acell with an agent that modulates one or more of the activities of humanphosphatidylserine synthase-like protein activity associated with thecell. An agent that modulates human phosphatidylserine synthase-likeprotein activity can be an agent as described herein, such as a nucleicacid or a protein, a naturally-occurring cognate ligand of a humanphosphatidylserine synthase-like protein, a peptide, a humanphosphatidylserine synthase-like peptidomimetic, or other smallmolecule. In one embodiment, the agent stimulates one or more of thebiological activities of human phosphatidylserine synthase-like protein.Examples of such stimulatory agents include active humanphosphatidylserine synthase-like protein and a nucleic acid moleculeencoding a human phosphatidylserine synthase-like protein that has beenintroduced into the cell. In another embodiment, the agent inhibits oneor more of the biological activities of human phosphatidylserinesynthase-like protein. Examples of such inhibitory agents includeantisense human phosphatidylserine synthase-like nucleic acid moleculesand anti-human phosphatidylserine synthase-like antibodies.

These modulatory methods can be performed in vitro (e.g., by culturingthe cell with the agent) or, alternatively, in vivo (e.g, byadministering the agent to a subject). As such, the present inventionprovides methods of treating an individual afflicted with a disease ordisorder characterized by aberrant expression or activity of a humanphosphatidylserine synthase-like protein or nucleic acid molecule. Inone embodiment, the method involves administering an agent (e.g., anagent identified by a screening assay described herein), or acombination of agents, that modulates (e.g., upregulates ordownregulates) human phosphatidylserine synthase-like expression oractivity. In another embodiment, the method involves administering ahuman phosphatidylserine synthase-like protein or nucleic acid moleculeas therapy to compensate for reduced or aberrant humanphosphatidylserine synthase-like expression or activity.

Stimulation of human phosphatidylserine synthase-like activity isdesirable in situations in which a human phosphatidylserinesynthase-like protein is abnormally downregulated and/or in whichincreased human phosphatidylserine synthase-like activity is likely tohave a beneficial effect. Conversely, inhibition of humanphosphatidylserine synthase-like activity is desirable in situations inwhich human phosphatidylserine synthase-like activity is abnormallyupregulated and/or in which decreased human phosphatidylserinesynthase-like activity is likely to have a beneficial effect.

This invention is further illustrated by the following examples, whichshould not be construed as limiting.

EXAMPLES Example 1 Identification and Characterization of Human 32670cDNAs

The human 32670 sequence (FIGS. 14A-B; SEQ ID NO:5), which isapproximately 1852 nucleotides long including untranslated regions,contains a predicted methionine-initiated coding sequence of about 1464nucleotides (nucleotides 14-1477 of SEQ ID NO:5; SEQ ID NO:7). Thecoding sequence encodes a 487 amino acid protein (SEQ ID NO:6).

Example 2 Tissue Distribution of 32670 mRNA

32670 expression was determined by the PCR in cDNA libraries generatedfrom various human tissues and cell types. 32670 expression wasdetectable in cDNA libraries generated from the following tissues:microvascular endothelial cells, umbilical vein endothelial cells, U937cells, CaCo cells, HeLa cells, fetal brain, bronchial epithelium,astrocytes, prostate epithelium, primary osteoblasts, keratinocytes,melanocytes, coronary smooth muscle cells, cerebellum, pituitary, aorticendothelial cells, fetal kidney, fetal liver, mengial, bone marrow,fetal thymus, fetal heart, mammary gland, tissue from a subject withcongestive heart failure, prostate smooth muscle cells, osteoblaststreated with LPS for 6 hours, fetal heart, tissue from a subject withBurkin's lymphoma, mammary epithelium, umbilical smooth muscle,bronchial smooth muscle, fetal spleen, esophagus, fetal liver, fetalskin, fetal adrenal gland, lung carcinoma tissue, A549 cells, fetaltestes, pulmonary artery smooth muscle, erythroleukemia cells, embryonickeratinocytes, tongue squamous cell carcinoma, fetal hypothalamus, CD3treated T cells, HPKII cells, testes, H160 cells, placenta, skeletalmuscle, kidney, HPK cells, uterus, 9 week fetus, salivary gland, testes,K563 cells, lung, thymus, skeletal muscle, prostate, Hep-G2 insulinomacells, normal breast epithelia, normal ovarian epithelia, normalmegakaryocytes, Th-2 induced T cell, fetal dorsal spinal cord, colon tolive metastasis, colon carcinoma, lung squamous cell carcinoma, d8dendritic cells, skin, ovarian ascites, IBD colon, dorsal root ganglia,brain subcortical white matter, prostate tumor xenograft cell cline K10,prostate cancer to liver metastasis, umbilical vein endothelial cellsgrown under hypoxic conditions, melanoma G361 cell line, lumbosacaralspinal chord, and adult bone marrow CD34 positive cells.

Northern blot hybridizations with various RNA samples are performedunder standard conditions and washed under stringent conditions, i.e.,0.2×SSC at 65° C. A DNA probe corresponding to all or a portion of the32670 cDNA (SEQ ID NO:5 or SEQ ID NO:7) can be used. The DNA isradioactively labeled with ³²P-dCTP using the Prime-It Kit (Stratagene,La Jolla, Calif.) according to the instructions of the supplier. Filterscontaining mRNA from mouse hematopoietic and endocrine tissues, andcancer cell lines (Clontech, Palo Alto, Calif.) are probed in ExpressHybhybridization solution (Clontech) and washed at high stringencyaccording to manufacturer's recommendations.

Example 3 Recombinant Expression of 32670 in Bacterial Cells

In this example, 32670 is expressed as a recombinantglutathione-S-transferase (GST) fusion polypeptide in E. coli and thefusion polypeptide is isolated and characterized. Specifically, 32670 isfused to GST and this fusion polypeptide is expressed in E. coli, e.g.,strain PEB199. Expression of the GST-32670 fusion protein in PEB199 isinduced with IPTG. The recombinant fusion polypeptide is purified fromcrude bacterial lysates of the induced PEB199 strain by affinitychromatography on glutathione beads. Using polyacrylamide gelelectrophoretic analysis of the polypeptide purified from the bacteriallysates, the molecular weight of the resultant fusion polypeptide isdetermined.

Example 4 Expression of Recombinant 32670 Protein in COS Cells

To express the 32670 gene in COS cells, the pcDNA/Amp vector byInvitrogen Corporation (San Diego, Calif.) is used. This vector containsan SV40 origin of replication, an ampicillin resistance gene, an E. colireplication origin, a CMV promoter followed by a polylinker region, andan SV40 intron and polyadenylation site. A DNA fragment encoding theentire 32670 protein and an HA tag (Wilson et al. (1984) Cell 37:767) ora FLAG tag fused in-frame to its 3′ end of the fragment is cloned intothe polylinker region of the vector, thereby placing the expression ofthe recombinant protein under the control of the CMV promoter.

To construct the plasmid, the 32670 DNA sequence is amplified by PCRusing two primers. The 5′ primer contains the restriction site ofinterest followed by approximately twenty nucleotides of the 32670coding sequence starting from the initiation codon; the 3′ end sequencecontains complementary sequences to the other restriction site ofinterest, a translation stop codon, the HA tag or FLAG tag and the last20 nucleotides of the 32670 coding sequence. The PCR amplified fragmentand the pCDNA/Amp vector are digested with the appropriate restrictionenzymes and the vector is dephosphorylated using the CIAP enzyme (NewEngland Biolabs, Beverly, Mass.). Preferably the two restriction siteschosen are different so that the 32670 gene is inserted in the correctorientation. The ligation mixture is transformed into E. coli cells(strains HB101, DH5α, SURE, available from Stratagene Cloning Systems,La Jolla, Calif., can be used), the transformed culture is plated onampicillin media plates, and resistant colonies are selected. PlasmidDNA is isolated from transformants and examined by restriction analysisfor the presence of the correct fragment.

COS cells are subsequently transfected with the 32670-pcDNA/Amp plasmidDNA using the calcium phosphate or calcium chloride co-precipitationmethods, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Other suitable methods for transfecting host cells canbe found in Sambrook, J., Fritsh, E. F., and Maniatis, T. MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Theexpression of the 32670 polypeptide is detected by radiolabelling(³⁵S-methionine or 35S-cysteine available from NEN, Boston, Mass., canbe used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1988) using an HA specific monoclonal antibody. Briefly,the cells are labeled for 8 hours with 35S-methionine (or 35S-cysteine).The culture media are then collected and the cells are lysed usingdetergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50mM Tris, pH 7.5). Both the cell lysate and the culture media areprecipitated with an HA specific monoclonal antibody. Precipitatedpolypeptides are then analyzed by SDS-PAGE.

Alternatively, DNA containing the 32670 coding sequence is cloneddirectly into the polylinker of the pCDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of the 32670polypeptide is detected by radiolabelling and immunoprecipitation usinga 32670 specific monoclonal antibody.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

Chapter 3 5698, A DNA Fragmentation Factor-Like Molecule and UsesThereof BACKGROUND OF THE INVENTION

Apoptosis is fundamentally important in a variety of physiological andpathological processes. Apoptotic cells undergo an orchestrated cascadeof events characterized by distinct morphological changes includingmembrane blebbing, cytoplasmic and nuclear degredation, chromatinaggregation, and formation of apoptotic bodies. A key molecular event inthe process of apoptosis is the activation of the caspase cascade.Caspases are a family of serine proteases identified in mammalian cells.Caspase activation leads to cleavage of target protein and execution ofthe apoptotic program. Apoptotic signals, including growth factor andinterleukin deprivation, activation of Fas, ionizing radiation, and aseries of chemicals acting as upstream signals, can convert theprecursors of caspases into the protease active enzymes.

One of the downstream substrates of caspase is a subunit of the DNAFragmentation Factor (DFF) complex. DFF is a heterodimeric proteincomplex composed of DFF45 and DFF40 subunits. DFF45 has been found to bethe substrate of caspase-3 and DFF40 has also been cloned and found tobe a DNA fragmentation nuclease. Studies have shown that DFF45 canmediate the correct folding of DFF40 and remains associated with DFF40to prevent DFF40 from being activated until a specific signal (theactivation of caspase-3) is received. DFF45 therefore acts as a specificmolecular chaperon and appears to provide a double safety control toprevent unwanted activation of DFF40. Following cleavage, the DFF45dissociates from DFF40. The active component of DFF then triggers bothDNA fragmentation and chromatin condensation during apoptosis (Gu et al.(1999) The Journal of Biological Chemistry 274:20759-20762).

DFF homologs have also been identified in the mouse. The mouse DFF iscomposed of three molecules: one caspase-activated DNAse (CAD) and twoforms of CAD inhibitors (ICAD-L and ICAD-S). Mouse CAD and ICAD-L areapparently the counterpart of huma DFF40 (CPAN) and DFF45, respectively,whereas the human counterpart of mouse ICAD-S has not been identified.

In addition, cell death-inducing DFF45-like effector A and B (CIDE-A andCIDE-B) encode highly related proteins with homology to the N-terminalregion of DFF45. CIDE-A and CIDE-B activate apoptosis and appear tofunction as positive effectors of the apoptotic pathway. Inohara et al.have demonstrated that CIDE-A and CIDE-B induce DNA fragmentation aswell as other morphological features of apoptosis including nuclearcondensation and membrane blebbing (Inohara et al. (1998) EMBO J.17:2526-2533).

The proteins of the DFF complex influence DNA fragmentation andultimately apoptosis and therefore play a role in various biological andpathological processes. For example, during normal CNS development, asignificant proportion of neurons die by apoptosis to permit thematching of cells with their targets (Oppenheim et al. (1991) Annu. Rev.Neuroscience 14: 453-501). Apoptotic events also play an important rolein specific pathological conditions including Alzheimer's andHuntington's disease (Portera-Calliau et al. (1995) J. Neurosci15:3775-3787 and Samle et al. (1995) Exp. Neruo. 133:225-230), cerebralischemia (MacManus et al. (1995) J. Cereb. Blood Flow Metab.15:728-737), and HIV encephalitis (Petito et al. (1995) Am. J. Pathol.146:1121-1130).

Furthermore, recent studies have demonstrated that apoptotic cell deathoccurs after traumatic spinal cord injury and following traumatic braininjury (Li et al. (1996) J. Neruopathol. Exp. Neruol 55:280-289). Theapoptotic event is characterized by the activation of caspase-3 in theinjured rat cortex and hippocampus. Regional and temporal changes inDFF-like proteins are observed following these trauma events. Zhang etal. observed that DFF45-like proteins labeled with the anti-human DFF45antibody significantly decrease in the cortex after brain trauma. Thesechanges in the DFF45-like proteins are suggestive of an early signal ofDNA fragmentation and that the DFF proteins are playing a role duringapoptosis following a traumatic brain injury (Zhang et al. (1999)Journal of Neurochemistry 73:1650-1659).

Evidence indicates DFF complex plays an important role in cellularapoptosis. Accordingly, it is valuable to the field of pharmaceuticaldevelopment to identify and characterize previously unknown DFF-likepolypeptides. The present invention advances the state of the art byproviding previously unidentified DNA fragmentation factor-like nucleicacid and polypeptides.

SUMMARY OF THE INVENTION

Isolated nucleic acid molecules corresponding to DFF-like nucleic acidsequences are provided. Additionally, amino acid sequences correspondingto the polynucleotides are encompassed. In particular, the presentinvention provides for isolated nucleic acid molecules comprisingnucleotide sequences encoding the amino acid sequences shown in SEQ IDNO:11. Further provided are DFF-like polypeptides having an amino acidsequence encoded by a nucleic acid molecule described herein.

The present invention also provides vectors and host cells forrecombinant expression of the nucleic acid molecules described herein,as well as methods of making such vectors and host cells and for usingthem for production of the polypeptides or peptides of the invention byrecombinant techniques.

The DFF-like molecules of the present invention are useful formodulating apoptotic events, including DNA fragmentation. The moleculesare useful for the diagnosis and treatment of disorders associated withdysregulated apoptosis. Such disorders include cancers, autoimmunedisorders, neurodegenerative diseases, ischemic injuries, and virusinduced lymphocyte depletion. Additionally, the molecules of theinvention are useful as modulating agents in a variety of cellularprocesses including DNA fragmentation, intracellular signaling, membraneblebbing, cytoplasmic and nuclear degradation, chromatin aggregation,and formation of apoptotic bodies. Accordingly, in one aspect, thisinvention provides isolated nucleic acid molecules encoding DFF-likeproteins or biologically active portions thereof, as well as nucleicacid fragments suitable as primers or hybridization probes for thedetection of DFF-like-encoding nucleic acids.

Another aspect of this invention features isolated or recombinantDFF-like proteins and polypeptides. Preferred DFF-like proteins andpolypeptides possess at least one biological activity possessed bynaturally occurring DFF-like proteins.

Variant nucleic acid molecules and polypeptides substantially homologousto the nucleotide and amino acid sequences set forth in the sequencelistings are encompassed by the present invention. Additionally,fragments and substantially homologous fragments of the nucleotide andamino acid sequences are provided.

Antibodies and antibody fragments that selectively bind the DFF-likepolypeptides and fragments are provided. Such antibodies are useful indetecting the DFF-like polypeptides as well as in regulating apoptoticprocesses.

In another aspect, the present invention provides a method for detectingthe presence of DFF-like activity or expression in a biological sampleby contacting the biological sample with an agent capable of detectingan indicator of DFF-like activity such that the presence of DFF-likeactivity is detected in the biological sample.

In yet another aspect, the invention provides a method for modulatingDFF-like activity comprising contacting a cell with an agent thatmodulates (inhibits or stimulates) DFF-like activity or expression suchthat DFF-like activity or expression in the cell is modulated. In oneembodiment, the agent is an antibody that specifically binds to DFF-likeprotein. In another embodiment, the agent modulates expression ofDFF-like protein by modulating transcription of a DFF-like gene,splicing of a DFF-like mRNA, or translation of a DFF-like mRNA. In yetanother embodiment, the agent is a nucleic acid molecule having anucleotide sequence that is antisense to the coding strand of theDFF-like mRNA or the DFF-like gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant DFF-likeprotein activity or nucleic acid expression by administering an agentthat is a DFF-like modulator to the subject. In one embodiment, theDFF-like modulator is a DFF-like protein. In another embodiment, theDFF-like modulator is a DFF-like nucleic acid molecule. In otherembodiments, the DFF-like modulator is a peptide, peptidomimetic, orother small molecule.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic lesion or mutation characterized byat least one of the following: (1) aberrant modification or mutation ofa gene encoding a DFF-like protein; (2) misregulation of a gene encodinga DFF-like protein; and (3) aberrant post-translational modification ofa DFF-like protein, wherein a wild-type form of the gene encodes aprotein with a DFF-like activity.

In another aspect, the invention provides a method for identifying acompound that binds to or modulates the activity of a DFF-like protein.In general, such methods entail measuring a biological activity of aDFF-like protein in the presence and absence of a test compound andidentifying those compounds that alter the activity of the DFF-likeprotein.

The invention also features methods for identifying a compound thatmodulates the expression of DFF-like genes by measuring the expressionof the DFF-like sequences in the presence and absence of the compound.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the invention are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

The present invention provides DFF-like molecules. By “DFF-likemolecules” is intended a novel human sequence referred to as 5698, andvariants and fragments thereof. These full-length gene sequences orfragments thereof are referred to as “DFF-like” sequences, indicatingthey share sequence similarity with DFF genes. Isolated nucleic acidmolecules comprising nucleotide sequences encoding the 5698 polypeptidewhose amino acid sequence is given in SEQ ID NO:11, or a variant orfragment thereof, are provided. A nucleotide sequence encoding the 5698polypeptide is set forth in SEQ ID NO:10 or 12. The sequences aremembers of the DNA Fragmentation Factor family.

The disclosed invention relates to methods and compositions for themodulation, diagnosis, and treatment of disorders associated withdysregulated apoptosis. By “disregulted apoptosis” is intended analteration in the apoptotic process that results in either aninappropirately low or high rate of apoptosis. Disorders associated withan inappropriately low rate of apoptosis may prolong survival ofabnormal cells. These accumulated cells can give rise to cancers,especially those carcinomas with p53 mutations, or homo-dependenttumors, such as breast, prostate, or ovarian cancers. Autoimmunedisorders also can arise if, for example, autoreactive lymphocytes arenot removed following an immune response. The molecules are also usefulfor the diagnosis and treatment of disorders associated with increasedapoptosis and excessive cell death. These disorders are characterized bya marked loss of normal or protective cells and include:neurodegenerative diseases, manifested by loss of specific sets ofneurons, such as in the spinal muscular atrophies; ischemic injuriessuch as in myocardial infarction and stroke; and, virus inducedlymphocyte depletion, such as in acquired immune deficiency syndrome.

The molecules are also useful for the diagnosis and treatment ofdisorders in tissues in which the transcript is expressed (see Example 1and FIGS. 20A-B). Disorders involving the heart, include but are notlimited to, heart failure, including but not limited to, cardiachypertrophy, left-sided heart failure, and right-sided heart failure;ischemic heart disease, including but not limited to angina pectoris,myocardial infarction, chronic ischemic heart disease, and suddencardiac death; hypertensive heart disease, including but not limited to,systemic (left-sided) hypertensive heart disease and pulmonary(right-sided) hypertensive heart disease; valvular heart disease,including but not limited to, valvular degeneration caused bycalcification, such as calcific aortic stenosis, calcification of acongenitally bicuspid aortic valve, and mitral annular calcification,and myxomatous degeneration of the mitral valve (mitral valve prolapse),rheumatic fever and rheumatic heart disease, infective endocarditis, andnoninfected vegetations, such as nonbacterial thrombotic endocarditisand endocarditis of systemic lupus erythematosus (Libman-Sacks disease),carcinoid heart disease, and complications of artificial valves;myocardial disease, including but not limited to dilated cardiomyopathy,hypertrophic cardiomyopathy, restrictive cardiomyopathy, andmyocarditis; pericardial disease, including but not limited to,pericardial effusion and hemopericardium and pericarditis, includingacute pericarditis and healed pericarditis, and rheumatoid heartdisease; neoplastic heart disease, including but not limited to, primarycardiac tumors, such as myxoma, lipoma, papillary fibroelastoma,rhabdomyoma, and sarcoma, and cardiac effects of noncardiac neoplasms;congenital heart disease, including but not limited to, left-to-rightshunts—late cyanosis, such as atrial septal defect, ventricular septaldefect, patent ductus arteriosus, and atrioventricular septal defect,right-to-left shunts—early cyanosis, such as tetralogy of fallot,transposition of great arteries, truncus arteriosus, tricuspid atresia,and total anomalous pulmonary venous connection, obstructive congenitalanomalies, such as coarctation of aorta, pulmonary stenosis and atresia,and aortic stenosis and atresia, and disorders involving cardiactransplantation.

Disorders involving the kidney include, but are not limited to,congenital anomalies including, but not limited to, cystic diseases ofthe kidney, that include but are not limited to, cystic renal dysplasia,autosomal dominant (adult) polycystic kidney disease, autosomalrecessive (childhood) polycystic kidney disease, and cystic diseases ofrenal medulla, which include, but are not limited to, medullary spongekidney, and nephronophthisis-uremic medullary cystic disease complex,acquired (dialysis-associated) cystic disease, such as simple cysts;glomerular diseases including pathologies of glomerular injury thatinclude, but are not limited to, in situ immune complex deposition, thatincludes, but is not limited to, anti-GBM nephritis, Heymann nephritis,and antibodies against planted antigens, circulating immune complexnephritis, antibodies to glomerular cells, cell-mediated immunity inglomerulonephritis, activation of alternative complement pathway,epithelial cell injury, and pathologies involving mediators ofglomerular injury including cellular and soluble mediators, acuteglomerulonephritis, such as acute proliferative (poststreptococcal,postinfectious) glomerulonephritis, including but not limited to,poststreptococcal glomerulonephritis and nonstreptococcal acuteglomerulonephritis, rapidly progressive (crescentic) glomerulonephritis,nephrotic syndrome, membranous glomerulonephritis (membranousnephropathy), minimal change disease (lipoid nephrosis), focal segmentalglomerulosclerosis, membranoproliferative glomerulonephritis, IgAnephropathy (Berger disease), focal proliferative and necrotizingglomerulonephritis (focal glomerulonephritis), hereditary nephritis,including but not limited to, Alport syndrome and thin membrane disease(benign familial hematuria), chronic glomerulonephritis, glomerularlesions associated with systemic disease, including but not limited to,systemic lupus erythematosus, Henoch-Schonlein purpura, bacterialendocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary andimmunotactoid glomerulonephritis, and other systemic disorders; diseasesaffecting tubules and interstitium, including acute tubular necrosis andtubulointerstitial nephritis, including but not limited to,pyelonephritis and urinary tract infection, acute pyelonephritis,chronic pyelonephritis and reflux nephropathy, and tubulointerstitialnephritis induced by drugs and toxins, including but not limited to,acute drug-induced interstitial nephritis, analgesic abuse nephropathy,nephropathy associated with nonsteroidal anti-inflammatory drugs, andother tubulointerstitial diseases including, but not limited to, uratenephropathy, hypercalcemia and nephrocalcinosis, and multiple myeloma;diseases of blood vessels including benign nephrosclerosis, malignanthypertension and accelerated nephrosclerosis, renal artery stenosis, andthrombotic microangiopathies including, but not limited to, classic(childhood) hemolytic-uremic syndrome, adult hemolytic-uremicsyndrome/thrombotic thrombocytopenic purpura, idiopathic HUS/TTP, andother vascular disorders including, but not limited to, atheroscleroticischemic renal disease, atheroembolic renal disease, sickle cell diseasenephropathy, diffuse cortical necrosis, and renal infarcts; urinarytract obstruction (obstructive uropathy); urolithiasis (renal calculi,stones); and tumors of the kidney including, but not limited to, benigntumors, such as renal papillary adenoma, renal fibroma or hamartoma(renomedullary interstitial cell tumor), angiomyolipoma, and oncocytoma,and malignant tumors, including renal cell carcinoma (hypernephroma,adenocarcinoma of kidney), which includes urothelial carcinomas of renalpelvis.

Disorders involving the skeletal muscle include tumors such asrhabdomyosarcoma.

Disorders involving the liver include, but are not limited to, hepaticinjury; jaundice and cholestasis, such as bilirubin and bile formation;hepatic failure and cirrhosis, such as cirrhosis, portal hypertension,including ascites, portosystemic shunts, and splenomegaly; infectiousdisorders, such as viral hepatitis, including hepatitis A-E infectionand infection by other hepatitis viruses, clinicopathologic syndromes,such as the carrier state, asymptomatic infection, acute viralhepatitis, chronic viral hepatitis, and fulminant hepatitis; autoimmunehepatitis; drug- and toxin-induced liver disease, such as alcoholicliver disease; inborn errors of metabolism and pediatric liver disease,such as hemochromatosis, Wilson disease, α₁-antitrypsin deficiency, andneonatal hepatitis; intrahepatic biliary tract disease, such assecondary biliary cirrhosis, primary biliary cirrhosis, primarysclerosing cholangitis, and anomalies of the biliary tree; circulatorydisorders, such as impaired blood flow into the liver, including hepaticartery compromise and portal vein obstruction and thrombosis, impairedblood flow through the liver, including passive congestion andcentrilobular necrosis and peliosis hepatis, hepatic vein outflowobstruction, including hepatic vein thrombosis (Budd-Chiari syndrome)and veno-occlusive disease; hepatic disease associated with pregnancy,such as preeclampsia and eclampsia, acute fatty liver of pregnancy, andintrehepatic cholestasis of pregnancy; hepatic complications of organ orbone marrow transplantation, such as drug toxicity after bone marrowtransplantation, graft-versus-host disease and liver rejection, andnonimmunologic damage to liver allografts; tumors and tumorousconditions, such as nodular hyperplasias, adenomas, and malignanttumors, including primary carcinoma of the liver and metastatic tumors.

The DFF-like sequences of the present invention find use in modulating aapoptosis. By “modulating” is intended the upregulating ordownregulating of a response. That is, the compositions of the inventionaffect the targeted activity in either a positive or negative fashion.The activation of apoptosis is manifested by changes including membraneblebbing, DNA fragmentation, cytoplasmic and nuclear degredation,chromatin aggregation, formation of apoptotic bodies, and cell death.

Proteins and/or antibodies of the invention are also useful inmodulating the apoptotic process.

The DFF-like gene, clone 5698 was identified in a human primaryosteoblast cDNA library. Clone 5698 encodes an mRNA transcript havingthe corresponding cDNA set forth in SEQ ID NO:10. This transcript has anucleotide open reading frame (nucleotides 169-828 of SEQ ID NO:10),which encodes a 219 amino acid protein (SEQ ID NO:11). Prosite programanalysis was used to predict various sites within the 5698 protein. AnN-glycosylation site was predicted at aa 18-21. Protein kinase Cphosphorylation sites were predicted at aa 46-48 and 199-201. Caseinkinase II phosphorylation sites were predicted at aa 55-58 and 82-85.N-myristoylation sites were predicted at aa 50-55 and 195-200. Anamidation site was predicted at aa 23-26. A leucine zipper pattern waspredicted at aa 179-200. The DFF-like protein possesses a CAD domain,from aa 36-108 as predicted by HMMer, Version 2. For general informationregarding PFAM identifiers, PS prefix and PF prefix domainidentification numbers, refer to Sonnhammer et al. (1997) Protein28:405-420 andhttp://www.psc.edu/general/software/packages/pfam/pfam.html.

As used herein, the term “CAD domain” includes an amino acid sequence ofabout 50-72 amino acid residues in length and having a bit score for thealignment of the sequence to the CAD domain (HMM) of at least 8.Preferably, an CAD domain includes at least about 1-72 amino acids,about 20-72 amino acid residues, or about 40-72 amino acids and has abit score for the alignment of the sequence to the CAD domain (HMM) ofat least 16 or greater(http://smart.embl-heidelberg.de/smart/selective.cgi?domains=cad&taxon_select=ALL&taxon_text=).An alignment of the CAD domain (amino acids 36 to 108 of SEQ ID NO:11)of the human DFF-like sequence of the invention with a consensus aminoacid sequence derived from a hidden Markov model is depicted in FIG. 17.

In a preferred embodiment a DFF-like polypeptide or protein has a “CADdomain” or a region which includes at least about 100-250 morepreferably about 130-200 or 160-200 amino acid residues and has at leastabout 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with an“CAD domain,” e.g., the CAD domain of a human DFF-like (e.g., amino acidresidues 36-108 of SEQ ID NO:11).

To identify the presence of an “CAD” domain in a DFF-like proteinsequence, and make the determination that a polypeptide or protein ofinterest has a particular profile, the amino acid sequence of theprotein can be searched against a database of HMMs (e.g., the Pfamdatabase, release 2.1) using the default parameters(http://www.sanger.ac.uk/Software/Pfam/HMM_search). For example, thehmmsf program, which is available as part of the HMMER package of searchprograms, is a family specific default program for MILPAT0063 and ascore of 15 is the default threshold score for determining a hit.Alternatively, the threshold score for determining a hit can be lowered(e.g., to 8 bits). A description of the Pfam database can be found inSonhammer et al. (1997) Proteins 28(3):405-420 and a detaileddescription of HMMs can be found, for example, in Gribskov et al. (1990)Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad.Sci. USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531;and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of whichare incorporated herein by reference.

The 5698 protein displays similarity to the Mus musculus cell deathactivator CIDE-B (SP Accession No. 3114594; Genbank Accession No.AAC34986; SEQ ID NO:14) and with the Homo sapiens cell death activatorCIDE-A (SP Accession No. 3114596; Genbank Accession No. AAC34987; SEQ IDNO:15). The sequence alignment was generated using the Clustal method.The 5698 protein shares approximately 83% identity with the murineCIDE-B and approximately 40% identity with the human CIDE-A amino acidsequence as determined by pairwise alignment.

The 5698 protein displays 37% identity from aa 36-209 to a ProDomconsensus sequence found in murine FSP27 (Genbank Accession No. P56198)and human CIDE-A (Genbank Accession No. 060543). FSP27 is an adipocytespecific protein that belongs to the DFF-45/ICAD family. FSP27 isassociated with terminal differentiation of fat cells and its expressionis regulated by the tumor necrosis pathway. See, for example, Danesch etal. (1992) J. Biol. Chem 267:7185-7193 and Williams et al. (1992) MolEndocrinol 6:1135-1141. CIDE-A has homology to the 45 kDa subunit of theDNA fragmentation factor. See, for example, Inohara et al. (1998) EmboJ. 17:2526-2533. The 5698 protein also displays 46% identity to a ProDomconsensus sequence found in the hypothetical protein F-121 of humanadenovirus type 2.

The DFF-like sequences of the invention are members of a family ofmolecules (the “DNA fragmentation factor-like”) having conservedfunctional features. The term “family” when referring to the proteinsand nucleic acid molecules of the invention is intended to mean two ormore proteins or nucleic acid molecules having sufficient amino acid ornucleotide sequence identity as defined herein. Such family members canbe naturally occurring and can be from either the same or differentspecies. For example, a family can contain a first protein of murineorigin and a homologue of that protein of human origin, as well as asecond, distinct protein of human origin and a murine homologue of thatprotein. Members of a family may also have common functionalcharacteristics.

Preferred DFF-like polypeptides of the present invention have an aminoacid sequence sufficiently identical to the amino acid sequence of SEQID NO:11. The term “sufficiently identical” is used herein to refer to afirst amino acid or nucleotide sequence that contains a sufficient orminimum number of identical or equivalent (e.g., with a similar sidechain) amino acid residues or nucleotides to a second amino acid ornucleotide sequence such that the first and second amino acid ornucleotide sequences have a common structural domain and/or commonfunctional activity. For example, amino acid or nucleotide sequencesthat contain a common structural domain having at least about 45%, 55%,or 65% identity, preferably 75% identity, more preferably 85%, 95%, or98% identity are defined herein as sufficiently identical.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. The percent identity between two sequences can bedetermined using techniques similar to those described below, with orwithout allowing gaps. In calculating percent identity, typically exactmatches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. In a preferred embodiment,the percent identity between two amino acid sequences is determinedusing the Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453 algorithmwhich has been incorporated into the GAP program in the GCG softwarepackage (available at http://www.gcg.com), using either a Blossum 62matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferredembodiment, the percent identity between two nucleotide sequences isdetermined using the GAP program in the GCG software package (availableat http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused if the practitioner is uncertain about what parameters should beapplied to determine if a molecule is within a sequence identity orhomology limitation of the invention) is using a Blossum 62 scoringmatrix with a gap open penalty of 12, a gap extend penalty of 4, and aframeshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of Karlin and Altschul (1990) Proc.Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al.(1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performedwith the NBLAST program, score=100, wordlength=12, to obtain nucleotidesequences homologous to DFF-like nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3, to obtain amino acid sequenceshomologous to DFF-like protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-Blast can be used to perform an iterated search thatdetects distant relationships between molecules. See Altschul et al.(1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blastprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Anotherpreferred, non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Myers and Miller (1988)CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program(version 2.0), which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

Accordingly, another embodiment of the invention features isolatedDFF-like proteins and polypeptides having a DFF-like protein activity.As used interchangeably herein, a “DFF-like protein activity”,“biological activity of a DFF-like protein”, or “functional activity ofa DFF-like protein” refers to an activity exerted by a DFF-like protein,polypeptide, or nucleic acid molecule on a DFF-like responsive cell asdetermined in vivo, or in vitro, according to standard assay techniques.A DFF-like activity can be a direct activity, such as an associationwith or an enzymatic activity on a second protein, or an indirectactivity, such as a cellular signaling activity mediated by interactionof the DFF-like protein with a second protein. In a preferredembodiment, a DFF-like activity includes at least one or more of thefollowing activities: (1) modulating (stimulating and/or enhancing orinhibiting) apoptotic events, including DNA fragmentation, membraneblebbing, cytoplasmic and nuclear degredation, chromatin aggregation,and formtion of apoptotic bodies (2) modulating the programmeddestruction of cells during embryogenesis including implantation,organogenesis, developmental involution and metamorphosis (3) modulatinghormone-dependent involution in the adult, such as endometrial cellbreakdown during the menstrual cycle, ovarian follicular atresia in themenopause, the regression of the lactating breast after weaning, andprostate atrophy after castration (4) modulating cell deletion inproliferating cell populations, such as intestinal crypt epithelia (5)modulating cell death in tumors (6) modulating the death of neutrophilsduring an acute inflammatory response (7) modulating the death of immunecells, both B and T lymphocytes after cytokine depletion (8) modulatingthe cell death induced by cytotoxic T-cells such as in cellular immunerejection and graft-verses-host diseases and (9) modulating atrophy inparenchymal organs after duct obstruction, such as occurs in thepancreas, parotid gland, and kidney.

An “isolated” or “purified” DFF-like nucleic acid molecule or protein,or biologically active portion thereof, is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Preferably, an “isolated” nucleicacid is free of sequences (preferably protein encoding sequences) thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For purposes of the invention,“isolated” when used to refer to nucleic acid molecules excludesisolated chromosomes. For example, in various embodiments, the isolatedDFF-like nucleic acid molecule can contain less than about 5 kb, 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturallyflank the nucleic acid molecule in genomic DNA of the cell from whichthe nucleic acid is derived. A DFF-like protein that is substantiallyfree of cellular material includes preparations of DFF-like proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofnon-DFF-like protein (also referred to herein as a “contaminatingprotein”). When the DFF-like protein or biologically active portionthereof is recombinantly produced, preferably, culture medium representsless than about 30%, 20%, 10%, or 5% of the volume of the proteinpreparation. When DFF-like protein is produced by chemical synthesis,preferably the protein preparations have less than about 30%, 20%, 10%,or 5% (by dry weight) of chemical precursors or non-DFF-like chemicals.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculescomprising nucleotide sequences encoding DFF-like proteins andpolypeptides or biologically active portions thereof, as well as nucleicacid molecules sufficient for use as hybridization probes to identifyDFF-like-encoding nucleic acids (e.g., DFF-like mRNA) and fragments foruse as PCR primers for the amplification or mutation of DFF-like nucleicacid molecules. As used herein, the term “nucleic acid molecule” isintended to include DNA molecules (e.g., cDNA or genomic DNA) and RNAmolecules (e.g., mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

Nucleotide sequences encoding the DFF-like proteins of the presentinvention include sequences set forth in SEQ ID NO:10, 12 andcomplements thereof. By “complement” is intended a nucleotide sequencethat is sufficiently complementary to a given nucleotide sequence suchthat it can hybridize to the given nucleotide sequence to thereby form astable duplex. The corresponding amino acid sequence for the DFF-likeprotein encoded by these nucleotide sequences is set forth in SEQ IDNO:11. The invention also encompasses nucleic acid molecules comprisingnucleotide sequences encoding partial-length DFF-like proteins,including the sequence set forth in SEQ ID NO:10 or 12, and complementsthereof.

Nucleic acid molecules that are fragments of these DFF-like nucleotidesequences are also encompassed by the present invention. By “fragment”is intended a portion of the nucleotide sequence encoding a DFF-likeprotein. A fragment of a DFF-like nucleotide sequence may encode abiologically active portion of a DFF-like protein, or it may be afragment that can be used as a hybridization probe or PCR primer usingmethods disclosed below. A biologically active portion of a DFF-likeprotein can be prepared by isolating a portion of one of the DFF-likenucleotide sequences of the invention, expressing the encoded portion ofthe DFF-like protein (e.g., by recombinant expression in vitro), andassessing the activity of the encoded portion of the DFF-like protein.Nucleic acid molecules that are fragments of a DFF-like nucleotidesequence comprise at least 15, 20, 50, 75, 100, 200, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400 nucleotides, or up to the number ofnucleotides present in a full-length DFF-like nucleotide sequencedisclosed herein (for example, 1284 nucleotides for SEQ ID NO:10)depending upon the intended use. Alternatively, a nucleic acid moleculesthat is a fragment of a DFF-like nucleotide sequence of the presentinvention comprises a nucleotide sequence consisting of nucleotides1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800,800-900, 1000-1100, 1100-1200, 1200-1284 of SEQ ID NO:10 or 12.

It is understood that isolated fragments include any contiguous sequencenot disclosed prior to the invention as well as sequences that aresubstantially the same and which are not disclosed. Accordingly, if anisolated fragment is disclosed prior to the present invention, thatfragment is not intended to be encompassed by the invention. When asequence is not disclosed prior to the present invention, an isolatednucleic acid fragment is at least about 12, 15, 20, 25, or 30 contiguousnucleotides. Other regions of the nucleotide sequence may comprisefragments of various sizes, depending upon potential homology withpreviously disclosed sequences.

A fragment of a DFF-like nucleotide sequence that encodes a biologicallyactive portion of a DFF-like protein of the invention will encode atleast 15, 25, 30, 50, 75, 100, 125, 150, 175, or 200 contiguous aminoacids, or up to the total number of amino acids present in a full-lengthDFF-like protein of the invention (for example, 219 amino acids for SEQID NO:11. Fragments of a DFF-like nucleotide sequence that are useful ashybridization probes for PCR primers generally need not encode abiologically active portion of a DFF-like protein.

Nucleic acid molecules that are variants of the DFF-like nucleotidesequences disclosed herein are also encompassed by the presentinvention. “Variants” of the DFF-like nucleotide sequences include thosesequences that encode the DFF-like proteins disclosed herein but thatdiffer conservatively because of the degeneracy of the genetic code.These naturally occurring allelic variants can be identified with theuse of well-known molecular biology techniques, such as polymerase chainreaction (PCR) and hybridization techniques as outlined below. Variantnucleotide sequences also include synthetically derived nucleotidesequences that have been generated, for example, by using site-directedmutagenesis but which still encode the DFF-like proteins disclosed inthe present invention as discussed below. Generally, nucleotide sequencevariants of the invention will have at least 45%, 55%, 65%, 75%, 85%,95%, or 98% identity to a particular nucleotide sequence disclosedherein. A variant DFF-like nucleotide sequence will encode a DFF-likeprotein that has an amino acid sequence having at least 45%, 55%, 65%,75%, 85%, 95%, or 98% identity to the amino acid sequence of a DFF-likeprotein disclosed herein.

In addition to the DFF-like nucleotide sequences shown in SEQ ID NOS:1and 3, it will be appreciated by those skilled in the art that DNAsequence polymorphisms that lead to changes in the amino acid sequencesof DFF-like proteins may exist within a population (e.g., the humanpopulation). Such genetic polymorphism in a DFF-like gene may existamong individuals within a population due to natural allelic variation.An allele is one of a group of genes that occur alternatively at a givengenetic locus. As used herein, the terms “gene” and “recombinant gene”refer to nucleic acid molecules comprising an open reading frameencoding a DFF-like protein, preferably a mammalian DFF-like protein. Asused herein, the phrase “allelic variant” refers to a nucleotidesequence that occurs at a DFF-like locus or to a polypeptide encoded bythe nucleotide sequence. Such natural allelic variations can typicallyresult in 1-5% variance in the nucleotide sequence of the DFF-like gene.Any and all such nucleotide variations and resulting amino acidpolymorphisms or variations in a DFF-like sequence that are the resultof natural allelic variation and that do not alter the functionalactivity of DFF-like proteins are intended to be within the scope of theinvention.

Moreover, nucleic acid molecules encoding DFF-like proteins from otherspecies (DFF-like homologues), which have a nucleotide sequencediffering from that of the DFF-like sequences disclosed herein, areintended to be within the scope of the invention. For example, nucleicacid molecules corresponding to natural allelic variants and homologuesof the human DFF-like cDNA of the invention can be isolated based ontheir identity to the human DFF-like nucleic acid disclosed herein usingthe human cDNA, or a portion thereof, as a hybridization probe accordingto standard hybridization techniques under stringent hybridizationconditions as disclosed below.

In addition to naturally-occurring allelic variants of the DFF-likesequences that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequences of the invention thereby leading to changes in theamino acid sequence of the encoded DFF-like proteins, without alteringthe biological activity of the DFF-like proteins. Thus, an isolatednucleic acid molecule encoding a DFF-like protein having a sequence thatdiffers from that of SEQ ID NO:11 can be created by introducing one ormore nucleotide substitutions, additions, or deletions into thecorresponding nucleotide sequence disclosed herein, such that one ormore amino acid substitutions, additions or deletions are introducedinto the encoded protein. Mutations can be introduced by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Such variant nucleotide sequences are also encompassed bythe present invention.

For example, preferably, conservative amino acid substitutions may bemade at one or more predicted, preferably nonessential amino acidresidues. A “nonessential” amino acid residue is a residue that can bealtered from the wild-type sequence of a DFF-like protein (e.g., thesequence of SEQ ID NO:11) without altering the biological activity,whereas an “essential” amino acid residue is required for biologicalactivity. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Such substitutions would not be made for conserved aminoacid residues, or for amino acid residues residing within a conservedmotif, such as the growth factor and cytokine receptor signature 2sequence and the U-PAR/Ly-6 domain sequence of SEQ ID NO:11, where suchresidues are essential for protein activity.

Alternatively, variant DFF-like nucleotide sequences can be made byintroducing mutations randomly along all or part of a DFF-like codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for DFF-like biological activity to identify mutantsthat retain activity. Following mutagenesis, the encoded protein can beexpressed recombinantly, and the activity of the protein can bedetermined using standard assay techniques.

Thus the nucleotide sequences of the invention include the sequencesdisclosed herein as well as fragments and variants thereof. The DFF-likenucleotide sequences of the invention, and fragments and variantsthereof, can be used as probes and/or primers to identify and/or cloneDFF-like homologues in other cell types, e.g., from other tissues, aswell as DFF-like homologues from other mammals. Such probes can be usedto detect transcripts or genomic sequences encoding the same oridentical proteins. These probes can be used as part of a diagnostictest kit for identifying cells or tissues that misexpress a DFF-likeprotein, such as by measuring levels of a DFF-like-encoding nucleic acidin a sample of cells from a subject, e.g., detecting DFF-like mRNAlevels or determining whether a genomic DFF-like gene has been mutatedor deleted.

In this manner, methods such as PCR, hybridization, and the like can beused to identify such sequences having substantial identity to thesequences of the invention. See, for example, Sambrook et al. (1989)Molecular Cloning: Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.) and Innis, et al. (1990) PCRProtocols: A Guide to Methods and Applications (Academic Press, NY).DFF-like nucleotide sequences isolated based on their sequence identityto the DFF-like nucleotide sequences set forth herein or to fragmentsand variants thereof are encompassed by the present invention.

In a hybridization method, all or part of a known DFF-like nucleotidesequence can be used to screen cDNA or genomic libraries. Methods forconstruction of such cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.). The so-called hybridization probes may be genomic DNAfragments, cDNA fragments, RNA fragments, or other oligonucleotides, andmay be labeled with a detectable group such as ³²P, or any otherdetectable marker, such as other radioisotopes, a fluorescent compound,an enzyme, or an enzyme co-factor. Probes for hybridization can be madeby labeling synthetic oligonucleotides based on the known DFF-likenucleotide sequence disclosed herein. Degenerate primers designed on thebasis of conserved nucleotides or amino acid residues in a knownDFF-like nucleotide sequence or encoded amino acid sequence canadditionally be used. The probe typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 50, 75, 100,125, 150, 175, 200, 250, 300, 350, or 400 consecutive nucleotides of aDFF-like nucleotide sequence of the invention or a fragment or variantthereof. Preparation of probes for hybridization is generally known inthe art and is disclosed in Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.), herein incorporated by reference.

For example, in one embodiment, a previously unidentified DFF-likenucleic acid molecule hybridizes under stringent conditions to a probethat is a nucleic acid molecule comprising one of the DFF-likenucleotide sequences of the invention or a fragment thereof. In anotherembodiment, the previously unknown DFF-like nucleic acid molecule is atleast 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800,900, 1000, 2,000, 3,000, 4,000 or 5,000 nucleotides in length andhybridizes under stringent conditions to a probe that is a nucleic acidmolecule comprising one of the DFF-like nucleotide sequences disclosedherein or a fragment thereof.

Accordingly, in another embodiment, an isolated previously unknownDFF-like nucleic acid molecule of the invention is at least 300, 325,350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1,100,1,200, 1,300, or 1,400 nucleotides in length and hybridizes understringent conditions to a probe that is a nucleic acid moleculecomprising one of the nucleotide sequences of the invention, preferablythe coding sequence set forth in SEQ ID NO:10, 12, or a complement,fragment, or variant thereof.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences typically remain hybridized to each other.Such stringent conditions are known to those skilled in the art and canbe found in Current Protocols in Molecular Biology (John Wiley & Sons,New York (1989)), 6.3.1-6.3.6. A preferred, example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50° C. Another example of stringent hybridizationconditions are hybridization in 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at55° C. A further example of stringent hybridization conditions arehybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.Preferably, stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 65° C. Particularly preferredstringency conditions (and the conditions that should be used if thepractitioner is uncertain about what conditions should be applied todetermine if a molecule is within a hybridization limitation of theinvention) are 0.5M Sodium Phosphate, 7% SDS at 65° C., followed by oneor more washes at 0.2×SSC, 1% SDS at 65° C. Preferably, an isolatednucleic acid molecule that hybridizes under stringent conditions to aDFF-like sequence of the invention corresponds to a naturally-occurringnucleic acid molecule. As used herein, a “naturally-occurring” nucleicacid molecule refers to an RNA or DNA molecule having a nucleotidesequence that occurs in nature (e.g., encodes a natural protein).

Thus, in addition to the DFF-like nucleotide sequences disclosed hereinand fragments and variants thereof, the isolated nucleic acid moleculesof the invention also encompass homologous DNA sequences identified andisolated from other cells and/or organisms by hybridization with entireor partial sequences obtained from the DFF-like nucleotide sequencesdisclosed herein or variants and fragments thereof.

The present invention also encompasses antisense nucleic acid molecules,i.e., molecules that are complementary to a sense nucleic acid encodinga protein, e.g., complementary to the coding strand of a double-strandedcDNA molecule, or complementary to an mRNA sequence. Accordingly, anantisense nucleic acid can hydrogen bond to a sense nucleic acid. Theantisense nucleic acid can be complementary to an entire DFF-like codingstrand, or to only a portion thereof, e.g., all or part of the proteincoding region (or open reading frame). An antisense nucleic acidmolecule can be antisense to a noncoding region of the coding strand ofa nucleotide sequence encoding a DFF-like protein. The noncoding regionsare the 5′ and 3′ sequences that flank the coding region and are nottranslated into amino acids.

Given the coding-strand sequence encoding a DFF-like protein disclosedherein (e.g., SEQ ID NO:10 or 12), antisense nucleic acids of theinvention can be designed according to the rules of Watson and Crickbase pairing. The antisense nucleic acid molecule can be complementaryto the entire coding region of DFF-like mRNA, but more preferably is anoligonucleotide that is antisense to only a portion of the coding ornoncoding region of DFF-like mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of DFF-like mRNA. An anti sense oligonucleotidecan be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50nucleotides in length. An antisense nucleic acid of the invention can beconstructed using chemical synthesis and enzymatic ligation proceduresknown in the art.

For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, including, but not limited to, for example e.g., phosphorothioatederivatives and acridine substituted nucleotides. Alternatively, theantisense nucleic acid can be produced biologically using an expressionvector into which a nucleic acid has been subcloned in an antisenseorientation (i.e., RNA transcribed from the inserted nucleic acid willbe of an antisense orientation to a target nucleic acid of interest,described further in the following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a DFF-likeprotein to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. An example of a route ofadministration of antisense nucleic acid molecules of the inventionincludes direct injection at a tissue site. Alternatively, antisensenucleic acid molecules can be modified to target selected cells and thenadministered systemically. For example, antisense molecules can belinked to peptides or antibodies to form a complex that specificallybinds to receptors or antigens expressed on a selected cell surface. Theantisense nucleic acid molecules can also be delivered to cells usingthe vectors described herein. To achieve sufficient intracellularconcentrations of the antisense molecules, vector constructs in whichthe antisense nucleic acid molecule is placed under the control of astrong pol II or pol III promoter are preferred.

An antisense nucleic acid molecule of the invention can be an a-anomericnucleic acid molecule. An a-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

The invention also encompasses ribozymes, which are catalytic RNAmolecules with ribonuclease activity that are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Ribozymes (e.g., hammerhead ribozymes (describedin Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used tocatalytically cleave DFF-like mRNA transcripts to thereby inhibittranslation of DFF-like mRNA. A ribozyme having specificity for aDFF-like-encoding nucleic acid can be designed based upon the nucleotidesequence of a DFF-like cDNA disclosed herein (e.g., SEQ ID NO:10 or 12).See, e.g., Cech et al., U.S. Pat. No. 4,987,071; and Cech et al., U.S.Pat. No. 5,116,742. Alternatively, DFF-like mRNA can be used to select acatalytic RNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel and Szostak (1993) Science 261:1411-1418.

The invention also encompasses nucleic acid molecules that form triplehelical structures. For example, DFF-like gene expression can beinhibited by targeting nucleotide sequences complementary to theregulatory region of the DFF-like protein (e.g., the DFF-like promoterand/or enhancers) to form triple helical structures that preventtranscription of the DFF-like gene in target cells. See generally Helene(1991) Anticancer Drug Des. 6(6):569; Helene (1992) Ann. N.Y. Acad. Sci.660:27; and Maher (1992) Bioassays 14(12):807.

In preferred embodiments, the nucleic acid molecules of the inventioncan be modified at the base moiety, sugar moiety, or phosphate backboneto improve, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal. (1996) Bioorganic & Medicinal Chemistry 4:5). As used herein, theterms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics,e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid-phase peptide synthesis protocols as described in Hyrupet al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci.USA 93:14670.

PNAs of a DFF-like molecule can be used in therapeutic and diagnosticapplications. For example, PNAs can be used as antisense or antigeneagents for sequence-specific modulation of gene expression by, e.g.,inducing transcription or translation arrest or inhibiting replication.PNAs of the invention can also be used, e.g., in the analysis of singlebase pair mutations in a gene by, e.g., PNA-directed PCR clamping; asartificial restriction enzymes when used in combination with otherenzymes, e.g., S1 nucleases (Hyrup (1996), supra; or as probes orprimers for DNA sequence and hybridization (Hyrup (1996), supra;Perry-O'Keefe et al. (1996), supra).

In another embodiment, PNAs of a DFF-like molecule can be modified,e.g., to enhance their stability, specificity, or cellular uptake, byattaching lipophilic or other helper groups to PNA, by the formation ofPNA-DNA chimeras, or by the use of liposomes or other techniques of drugdelivery known in the art. The synthesis of PNA-DNA chimeras can beperformed as described in Hyrup (1996), supra; Finn et al. (1996)Nucleic Acids Res. 24(17):3357-63; Mag et al. (1989) Nucleic Acids Res.17:5973; and Peterson et al. (1975) Bioorganic Med. Chem. Lett. 5:1119.

II. Isolated DFF-Like Proteins and Anti-DFF Antibodies

DFF-like proteins are also encompassed within the present invention. By“DFF-like protein” is intended a protein having the amino acid sequenceset forth in SEQ ID NO:11, as well as fragments, biologically activeportions, and variants thereof.

“Fragments” or “biologically active portions” include polypeptidefragments suitable for use as immunogens to raise anti-DFF-likeantibodies. Fragments include peptides comprising amino acid sequencessufficiently identical to or derived from the amino acid sequence of aDFF-like protein, or partial-length protein, of the invention andexhibiting at least one activity of a DFF-like protein, but whichinclude fewer amino acids than the full-length (SEQ ID NO:11) DFF-likeprotein disclosed herein. Typically, biologically active portionscomprise a domain or motif with at least one activity of the DFF-likeprotein. A biologically active portion of a DFF-like protein can be apolypeptide which is, for example, 10, 25, 50, 100 or more amino acidsin length. Alternatively, a fragment of a polypeptide of the presentinvention comprises an amino acid sequence consisting of amino acidresidues 1-20, 20-40, 40-60, 60-80, 80-100, 100-120, 120-140, 140-160,160-180, 180-200, 200-219 of SEQ ID NO:11. Such biologically activeportions can be prepared by recombinant techniques and evaluated for oneor more of the functional activities of a native DFF-like protein. Asused here, a fragment comprises at least 5 contiguous amino acids of SEQID NO:11. The invention encompasses other fragments, however, such asany fragment in the protein greater than 6, 7, 8, or 9 amino acids.

By “variants” is intended proteins or polypeptides having an amino acidsequence that is at least about 45%, 55%, 65%, preferably about 75%,85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:11.Variants also include polypeptides encoded by a nucleic acid moleculethat hybridizes to the nucleic acid molecule of SEQ ID NOS:1 or 3, or acomplement thereof, under stringent conditions. In another embodiment, avariant of an isolated polypeptide of the present invention differs, byat least 1, but less than 5, 10, 20, 50, or 100 amino acid residues fromthe sequence shown in SEQ ID NO:11. If alignment is needed for thiscomparison the sequences should be aligned for maximum identity.“Looped” out sequences from deletions or insertions, or mismatches, areconsidered differences. Such variants generally retain the functionalactivity of the DFF-like proteins of the invention. Variants includepolypeptides that differ in amino acid sequence due to natural allelicvariation or mutagenesis.

The invention also provides DFF-like chimeric or fusion proteins. Asused herein, a DFF-like “chimeric protein” or “fusion protein” comprisesa DFF-like polypeptide operably linked to a non-DFF-like polypeptide. A“DFF-like polypeptide” refers to a polypeptide having an amino acidsequence corresponding to a DFF protein, whereas a “non-DFF-likepolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to a protein that is not substantially identical to theDFF-like protein, e.g., a protein that is different from the DFF-likeprotein and which is derived from the same or a different organism.Within a DFF-like fusion protein, the DFF-like polypeptide cancorrespond to all or a portion of a DFF-like protein, preferably atleast one biologically active portion of a DFF-like protein. Within thefusion protein, the term “operably linked” is intended to indicate thatthe DFF-like polypeptide and the non-DFF-like polypeptide are fusedin-frame to each other. The non-DFF-like polypeptide can be fused to theN-terminus or C-terminus of the DFF-like polypeptide.

One useful fusion protein is a GST-DFF-like fusion protein in which theDFF-like sequences are fused to the C-terminus of the GST sequences.Such fusion proteins can facilitate the purification of recombinantDFF-like proteins.

In yet another embodiment, the fusion protein is aDFF-like-immunoglobulin fusion protein in which all or part of aDFF-like protein is fused to sequences derived from a member of theimmunoglobulin protein family. The DFF-like-immunoglobulin fusionproteins of the invention can be incorporated into pharmaceuticalcompositions and administered to a subject to inhibit an interactionbetween a DFF-like ligand and a DFF-like protein on the surface of acell, thereby suppressing DFF-like-mediated signal transduction in vivo.The DFF-like-immunoglobulin fusion proteins can be used to affect thebioavailability of a DFF-like cognate ligand. Inhibition of the DFF-likeligand/DFF-like interaction may be useful therapeutically, both fortreating proliferative and differentiative disorders and for modulating(e.g., promoting or inhibiting) cell survival. Moreover, theDFF-like-immunoglobulin fusion proteins of the invention can be used asimmunogens to produce anti-DFF-like antibodies in a subject, to purifyDFF-like ligands, and in screening assays to identify molecules thatinhibit the interaction of a DFF-like protein with a DFF-like ligand.

Preferably, a DFF-like chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences may be ligatedtogether in-frame, or the fusion gene can be synthesized, such as withautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers that give rise tocomplementary overhangs between two consecutive gene fragments, whichcan subsequently be annealed and reamplified to generate a chimeric genesequence (see, e.g., Ausubel et al., eds. (1995) Current Protocols inMolecular Biology) (Greene Publishing and Wiley-Interscience, NY).Moreover, a DFF-like-encoding nucleic acid can be cloned into acommercially available expression vector such that it is linked in-frameto an existing fusion moiety.

Variants of the DFF-like proteins can function as either DFF-likeagonists (mimetics) or as DFF-like antagonists. Variants of the DFF-likeprotein can be generated by mutagenesis, e.g., discrete point mutationor truncation of the DFF-like protein. An agonist of the DFF-likeprotein can retain substantially the same, or a subset, of thebiological activities of the naturally occurring form of the DFF-likeprotein. An antagonist of the DFF-like protein can inhibit one or moreof the activities of the naturally occurring form of the DFF-likeprotein by, for example, competitively binding to a downstream orupstream member of a cellular signaling cascade that includes theDFF-like protein. Thus, specific biological effects can be elicited bytreatment with a variant of limited function. Treatment of a subjectwith a variant having a subset of the biological activities of thenaturally occurring form of the protein can have fewer side effects in asubject relative to treatment with the naturally occurring form of theDFF-like proteins.

Variants of a DFF-like protein that function as either DFF-like agonistsor as DFF-like antagonists can be identified by screening combinatoriallibraries of mutants, e.g., truncation mutants, of a DFF-like proteinfor DFF-like protein agonist or antagonist activity. In one embodiment,a variegated library of DFF-like variants is generated by combinatorialmutagenesis at the nucleic acid level and is encoded by a variegatedgene library. A variegated library of DFF-like variants can be producedby, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential DFF-like sequences is expressible as individual polypeptides,or alternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of DFF-like sequences therein. There are avariety of methods that can be used to produce libraries of potentialDFF-like variants from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential DFF-like sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidRes. 11:477).

In addition, libraries of fragments of a DFF-like protein codingsequence can be used to generate a variegated population of DFF-likefragments for screening and subsequent selection of variants of aDFF-like protein. In one embodiment, a library of coding sequencefragments can be generated by treating a double-stranded PCR fragment ofa DFF-like coding sequence with a nuclease under conditions whereinnicking occurs only about once per molecule, denaturing thedouble-stranded DNA, renaturing the DNA to form double-stranded DNAwhich can include sense/antisense pairs from different nicked products,removing single-stranded portions from reformed duplexes by treatmentwith S1 nuclease, and ligating the resulting fragment library into anexpression vector. By this method, one can derive an expression librarythat encodes N-terminal and internal fragments of various sizes of theDFF-like protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of DFF-like proteins. Themost widely used techniques, which are amenable to high through-putanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquethat enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify DFF-likevariants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).

An isolated DFF-like polypeptide of the invention can be used as animmunogen to generate antibodies that bind DFF-like proteins usingstandard techniques for polyclonal and monoclonal antibody preparation.The full-length DFF-like protein can be used or, alternatively, theinvention provides antigenic peptide fragments of DFF-like proteins foruse as immunogens. The antigenic peptide of a DFF-like protein comprisesat least 8, preferably 10, 15, 20, or 30 amino acid residues of theamino acid sequence shown in SEQ ID NO:11 and encompasses an epitope ofa DFF-like protein such that an antibody raised against the peptideforms a specific immune complex with the DFF-like protein. Preferredepitopes encompassed by the antigenic peptide are regions of a DFF-likeprotein that are located on the surface of the protein, e.g.,hydrophilic regions.

Accordingly, another aspect of the invention pertains to anti-DFF-likepolyclonal and monoclonal antibodies that bind a DFF-like protein.Polyclonal anti-DFF-like antibodies can be prepared by immunizing asuitable subject (e.g., rabbit, goat, mouse, or other mammal) with aDFF-like immunogen. The anti-DFF-like antibody titer in the immunizedsubject can be monitored over time by standard techniques, such as withan enzyme linked immunosorbent assay (ELISA) using immobilized DFF-likeprotein. At an appropriate time after immunization, e.g., when theanti-DFF-like antibody titers are highest, antibody-producing cells canbe obtained from the subject and used to prepare monoclonal antibodiesby standard techniques, such as the hybridoma technique originallydescribed by Kohler and Milstein (1975) Nature 256:495-497, the human Bcell hybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), theEBV-hybridoma technique (Cole et al. (1985) in Monoclonal Antibodies andCancer Therapy, ed. Reisfeld and Sell (Alan R. Liss, Inc., New York,N.Y.), pp. 77-96) or trioma techniques. The technology for producinghybridomas is well known (see generally Coligan et al., eds. (1994)Current Protocols in Immunology (John Wiley & Sons, Inc., New York,N.Y.); Galfre et al. (1977) Nature 266:550-52; Kenneth (1980) inMonoclonal Antibodies: A New Dimension In Biological Analyses (PlenumPublishing Corp., NY; and Lerner (1981) Yale J. Biol. Med., 54:387-402).

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-DFF-like antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with a DFF-like protein to therebyisolate immunoglobulin library members that bind the DFF-like protein.Kits for generating and screening phage display libraries arecommercially available (e.g., the Pharmacia Recombinant Phage AntibodySystem, Catalog No. 27-9400-01; and the Stratagene SufZAP™ Phage DisplayKit, Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, U.S. Pat. No. 5,223,409;PCT Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679;93/01288; WO 92/01047; 92/09690; and 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J. 12:725-734.

Additionally, recombinant anti-DFF-like antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and nonhumanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in PCT PublicationNos. WO 86101533 and WO 87/02671; European Patent Application Nos.184,187, 171, 496, 125,023, and 173,494; U.S. Pat. Nos. 4,816,567 and5,225,539; European Patent Application 125,023; Better et al. (1988)Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987)Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw etal. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison (1985) Science229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice that are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. See, for example, Lonberg and Huszar (1995)Int. Rev. Immunol. 13:65-93); and U.S. Pat. Nos. 5,625,126; 5,633,425;5,569,825; 5,661,016; and 5,545,806. In addition, companies such asAbgenix, Inc. (Freemont, Calif.), can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. This technology is described by Jespers etal. (1994) Bio/Technology 12:899-903).

An anti-DFF-like antibody (e.g., monoclonal antibody) can be used toisolate DFF-like proteins by standard techniques, such as affinitychromatography or immunoprecipitation. An anti-DFF-like antibody canfacilitate the purification of natural DFF-like protein from cells andof recombinantly produced DFF-like protein expressed in host cells.Moreover, an anti-DFF-like antibody can be used to detect DFF-likeprotein (e.g., in a cellular lysate or cell supernatant) in order toevaluate the abundance and pattern of expression of the DFF-likeprotein. Anti-DFF-like antibodies can be used diagnostically to monitorprotein levels in tissue as part of a clinical testing procedure, e.g.,to, for example, determine the efficacy of a given treatment regimen.Detection can be facilitated by coupling the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S,or ³H.

Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). The conjugates of the invention canbe used for modifying a given biological response, the drug moiety isnot to be construed as limited to classical chemical therapeutic agents.For example, the drug moiety may be a protein or polypeptide possessinga desired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, alpha-interferon,beta-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a DFF-likeprotein (or a portion thereof). “Vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked, such as a “plasmid”, a circular double-stranded DNA loopinto which additional DNA segments can be ligated, or a viral vector,where additional DNA segments can be ligated into the viral genome. Thevectors are useful for autonomous replication in a host cell or may beintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome (e.g.,nonepisomal mammalian vectors). Expression vectors are capable ofdirecting the expression of genes to which they are operably linked. Ingeneral, expression vectors of utility in recombinant DNA techniques areoften in the form of plasmids (vectors). However, the invention isintended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenoviruses,and adeno-associated viruses), that serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell. This means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, operably linked to the nucleicacid sequence to be expressed. “Operably linked” is intended to meanthat the nucleotide sequence of interest is linked to the regulatorysequence(s) in a manner that allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in ahost cell when the vector is introduced into the host cell). The term“regulatory sequence” is intended to include promoters, enhancers, andother expression control elements (e.g., polyadenylation signals). See,for example, Goeddel (1990) in Gene Expression Technology: Methods inEnzymology 185 (Academic Press, San Diego, Calif.). Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cell and those that direct expression ofthe nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., DFF-like proteins, mutant formsof DFF-like proteins, fusion proteins, etc.).

It is further recognized that the nucleic acid sequences of theinvention can be altered to contain codons, which are preferred, or nonpreferred, for a particular expression system. For example, the nucleicacid can be one in which at least one altered codon, and preferably atleast 10%, or 20% of the codons have been altered such that the sequenceis optimized for expression in E. coli, yeast, human, insect, or CHOcells. Methods for determining such codon usage are well known in theart.

The recombinant expression vectors of the invention can be designed forexpression of DFF-like protein in prokaryotic or eukaryotic host cells.Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or nonfusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Typical fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly,Mass.), and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein. Examples of suitableinducible nonfusion E. coli expression vectors include pTrc (Amann etal. (1988) Gene 69:301-315) and pET 11d (Studier et al. (1990) in GeneExpression Technology: Methods in Enzymology 185 (Academic Press, SanDiego, Calif.), pp. 60-89). Strategies to maximize recombinant proteinexpression in E. coli can be found in Gottesman (1990) in GeneExpression Technology: Methods in Enzymology 185 (Academic Press, CA),pp. 119-128 and Wada et al. (1992) Nucleic Acids Res. 20:2111-2118.Target gene expression from the pTrc vector relies on host RNApolymerase transcription from a hybrid trp-lac fusion promoter.

Suitable eukaryotic host cells include insect cells (examples ofBaculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39)); yeast cells (examples of vectorsfor expression in yeast S. cereivisiae include pYepSec1 (Baldari et al.(1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2(Invitrogen Corporation, San Diego, Calif.), and pPicZ (InvitrogenCorporation, San Diego, Calif.)); or mammalian cells (mammalianexpression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC(Kaufman et al. (1987) EMBO J. 6:187:195)). Suitable mammalian cellsinclude Chinese hamster ovary cells (CHO) or COS cells. In mammaliancells, the expression vector's control functions are often provided byviral regulatory elements. For example, commonly used promoters arederived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus40. For other suitable expression systems for both prokaryotic andeukaryotic cells, see chapters 16 and 17 of Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.). See, Goeddel (1990) in GeneExpression Technology: Methods in Enzymology 185 (Academic Press, SanDiego, Calif.). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell but are stillincluded within the scope of the term as used herein. A “purifiedpreparation of cells”, as used herein, refers to, in the case of plantor animal cells, an in vitro preparation of cells and not an entireintact plant or animal. In the case of cultured cells or microbialcells, it consists of a preparation of at least 10% and more preferably50% of the subject cells.

In one embodiment, the expression vector is a recombinant mammalianexpression vector that comprises tissue-specific regulatory elementsthat direct expression of the nucleic acid preferentially in aparticular cell type. Suitable tissue-specific promoters include thealbumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev.1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv.Immunol. 43:235-275), in particular promoters of T cell receptors(Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins(Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell33:741-748), neuron-specific promoters (e.g., the neurofilamentpromoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science230:912-916), and mammary gland-specific promoters (e.g., milk wheypromoter; U.S. Pat. No. 4,873,316 and European Application PatentPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379), the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546), and the like.

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperably linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to DFF-like mRNA. Regulatory sequences operably linkedto a nucleic acid cloned in the antisense orientation can be chosen todirect the continuous expression of the antisense RNA molecule in avariety of cell types, for instance viral promoters and/or enhancers, orregulatory sequences can be chosen to direct constitutive,tissue-specific, or cell-type-specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid, or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub et al. (1986)Reviews—Trends in Genetics, Vol. 1(1).

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.) and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin, and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding a DFF-like protein or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) DFF-likeprotein. Accordingly, the invention further provides methods forproducing DFF-like protein using the host cells of the invention. In oneembodiment, the method comprises culturing the host cell of theinvention, into which a recombinant expression vector encoding aDFF-like protein has been introduced, in a suitable medium such thatDFF-like protein is produced. In another embodiment, the method furthercomprises isolating DFF-like protein from the medium or the host cell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichDFF-like-coding sequences have been introduced. Such host cells can thenbe used to create nonhuman transgenic animals in which exogenousDFF-like sequences have been introduced into their genome or homologousrecombinant animals in which endogenous DFF-like sequences have beenaltered. Such animals are useful for studying the function and/oractivity of DFF-like genes and proteins and for identifying and/orevaluating modulators of DFF-like activity. As used herein, a“transgenic animal” is a nonhuman animal, preferably a mammal, morepreferably a rodent such as a rat or mouse, in which one or more of thecells of the animal includes a transgene. Other examples of transgenicanimals include nonhuman primates, sheep, dogs, cows, goats, chickens,amphibians, etc. A transgene is exogenous DNA that is integrated intothe genome of a cell from which a transgenic animal develops and whichremains in the genome of the mature animal, thereby directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic animal. As used herein, a “homologousrecombinant animal” is a nonhuman animal, preferably a mammal, morepreferably a mouse, in which an endogenous DFF-like gene has beenaltered by homologous recombination between the endogenous gene and anexogenous DNA molecule introduced into a cell of the animal, e.g., anembryonic cell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducingDFF-like-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The DFF-likecDNA sequence can be introduced as a transgene into the genome of anonhuman animal. Alternatively, a homologue of the mouse DFF-like genecan be isolated based on hybridization and used as a transgene. Intronicsequences and polyadenylation signals can also be included in thetransgene to increase the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to theDFF-like transgene to direct expression of DFF-like protein toparticular cells. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866, 4,870,009, and 4,873,191 and in Hogan (1986)Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1986). Similar methods are used for production ofother transgenic animals. A transgenic founder animal can be identifiedbased upon the presence of the DFF-like transgene in its genome and/orexpression of DFF-like mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding DFF-like gene can further be bred to other transgenicanimals carrying other transgenes.

To create a homologous recombinant animal, one prepares a vectorcontaining at least a portion of a DFF-like gene or a homolog of thegene into which a deletion, addition, or substitution has beenintroduced to thereby alter, e.g., functionally disrupt, the DFF-likegene. In a preferred embodiment, the vector is designed such that, uponhomologous recombination, the endogenous DFF-like gene is functionallydisrupted (i.e., no longer encodes a functional protein; also referredto as a “knock out” vector). Alternatively, the vector can be designedsuch that, upon homologous recombination, the endogenous DFF-like geneis mutated or otherwise altered but still encodes functional protein(e.g., the upstream regulatory region can be altered to thereby alterthe expression of the endogenous DFF-like protein). In the homologousrecombination vector, the altered portion of the DFF-like gene isflanked at its 5′ and 3′ ends by additional nucleic acid of the DFF-likegene to allow for homologous recombination to occur between theexogenous DFF-like gene carried by the vector and an endogenous DFF-likegene in an embryonic stem cell. The additional flanking DFF-like nucleicacid is of sufficient length for successful homologous recombinationwith the endogenous gene. Typically, several kilobases of flanking DNA(both at the 5′ and 3′ ends) are included in the vector (see, e.g.,Thomas and Capecchi (1987) Cell 51:503 for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation), and cells in which the introducedDFF-like gene has homologously recombined with the endogenous DFF-likegene are selected (see, e.g., Li et al. (1992) Cell 69:915). Theselected cells are then injected into a blastocyst of an animal (e.g., amouse) to form aggregation chimeras (see, e.g., Bradley (1987) inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach, ed.Robertson (IRL, Oxford pp. 113-152). A chimeric embryo can then beimplanted into a suitable pseudopregnant female foster animal and theembryo brought to term. Progeny harboring the homologously recombinedDNA in their germ cells can be used to breed animals in which all cellsof the animal contain the homologously recombined DNA by germlinetransmission of the transgene. Methods for constructing homologousrecombination vectors and homologous recombinant animals are describedfurther in Bradley (1991) Current Opinion in Bio/Technology 2:823-829and in PCT Publication Nos. WO 90/11354, WO 91/01140, WO 92/0968, and WO93/04169.

In another embodiment, transgenic nonhuman animals containing selectedsystems that allow for regulated expression of the transgene can beproduced. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355). If a cre/oxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the nonhuman transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and PCT Publication Nos. WO 97/07668 and WO 97/07669.

IV. Pharmaceutical Compositions

The DFF-like nucleic acid molecules, DFF-like proteins, andanti-DFF-like antibodies (also referred to herein as “active compounds”)of the invention can be incorporated into pharmaceutical compositionssuitable for administration. Such compositions typically comprise thenucleic acid molecule, protein, or antibody and a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The compositions of the invention are useful to treat any of thedisorders discussed herein. The compositions are provided intherapeutically effective amounts. By “therapeutically effectiveamounts” is intended an amount sufficient to modulate the desiredresponse. As defined herein, a therapeutically effective amount ofprotein or polypeptide (i.e., an effective dosage) ranges from about0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg bodyweight, more preferably about 0.1 to 20 mg/kg body weight, and even morepreferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7mg/kg, or 5 to 6 mg/kg body weight.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of a protein, polypeptide, or antibody can include asingle treatment or, preferably, can include a series of treatments. Ina preferred example, a subject is treated with antibody, protein, orpolypeptide in the range of between about 0.1 to 20 mg/kg body weight,one time per week for between about 1 to 10 weeks, preferably between 2to 8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody, protein, or polypeptide used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays as described herein.

The present invention encompasses agents which modulate expression oractivity. An agent may, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e., including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds.

It is understood that appropriate doses of small molecule agents dependsupon a number of factors within the knowledge of the ordinarily skilledphysician, veterinarian, or researcher. The dose(s) of the smallmolecule will vary, for example, depending upon the identity, size, andcondition of the subject or sample being treated, further depending uponthe route by which the composition is to be administered, if applicable,and the effect which the practitioner desires the small molecule to haveupon the nucleic acid or polypeptide of the invention. Exemplary dosesinclude milligram or microgram amounts of the small molecule perkilogram of subject or sample weight (e.g., about 1 microgram perkilogram to about 500 milligrams per kilogram, about 100 micrograms perkilogram to about 5 milligrams per kilogram, or about 1 microgram perkilogram to about 50 micrograms per kilogram. It is furthermoreunderstood that appropriate doses of a small molecule depend upon thepotency of the small molecule with respect to the expression or activityto be modulated. Such appropriate doses may be determined using theassays described herein. When one or more of these small molecules is tobe administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes, or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF; Parsippany, N.J.), or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion, and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a DFF-like protein or anti-DFF-like antibody) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying, which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth, or gelatin; an excipientsuch as starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. For administrationby inhalation, the compounds are delivered in the form of an aerosolspray from a pressurized container or dispenser that contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art. Thecompounds can also be prepared in the form of suppositories (e.g., withconventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated with each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. Depending on thetype and severity of the disease, about 1 μg/kg to about 15 mg/kg (e.g.,0.1 to 20 mg/kg) of antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to about 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays. Anexemplary dosing regimen is disclosed in WO 94/04188. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470), or by stereotactic injection(see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods:(a) screening assays; (b) detection assays (e.g., chromosomal mapping,tissue typing, forensic biology); (c) predictive medicine (e.g.,diagnostic assays, prognostic assays, monitoring clinical trials, andpharmacogenomics); and (d) methods of treatment (e.g., therapeutic andprophylactic). The isolated nucleic acid molecules of the invention canbe used to express DFF-like protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect DFF-likemRNA (e.g., in a biological sample) or a genetic lesion in a DFF-likegene, and to modulate DFF-like activity. In addition, the DFF-likeproteins can be used to screen drugs or compounds that modulateapoptotic events as well as to treat disorders characterized byinsufficient or excessive production of DFF-like protein or productionof DFF-like protein forms that have decreased or aberrant activitycompared to DFF-like wild type protein. In addition, the anti-DFF-likeantibodies of the invention can be used to detect and isolate DFF-likeproteins and modulate DFF-like activity.

A. Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules, or otherdrugs) that bind to DFF-like proteins or have a stimulatory orinhibitory effect on, for example, DFF-like expression or DFF-likeactivity.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including biological libraries, spatially addressable parallelsolid phase or solution phase libraries, synthetic library methodsrequiring deconvolution, the “one-bead one-compound” library method, andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, nonpeptide oligomer, orsmall molecule libraries of compounds (Lam (1997) Anticancer Drug Des.12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andGallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Bio/Techniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Pat.No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA89:1865-1869), or phage (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. USA 87:6378-6382; and Felici (1991) J. Mol. Biol.222:301-310).

Determining the ability of the test compound to bind to the DFF-likeprotein can be accomplished, for example, by coupling the test compoundwith a radioisotope or enzymatic label such that binding of the testcompound to the DFF-like protein or biologically active portion thereofcan be determined by detecting the labeled compound in a complex. Forexample, test compounds can be labeled with ¹²⁵I, ³⁵S, 14C, or ³H,either directly or indirectly, and the radioisotope detected by directcounting of radioemission or by scintillation counting. Alternatively,test compounds can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product.

In a similar manner, one may determine the ability of the DFF-likeprotein to bind to or interact with a DFF-like target molecule. By“target molecule” is intended a molecule with which a DFF-like proteinbinds or interacts in nature. In a preferred embodiment, the ability ofthe DFF-like protein to bind to or interact with a DFF-like targetmolecule can be determined by monitoring the activity of the targetmolecule. For example, the activity of the target molecule can bemonitored by detecting a morphological change induced by apoptosis(e.g., DNA fragmentation, membrane blebbing, cytoplasmic and nucleardegradation, etc.), detecting catalytic/enzymatic activity of the targeton an appropriate substrate, or detecting the induction of a reportergene (e.g., DFF-like-responsive regulatory element operably linked to anucleic acid encoding a detectable marker, e.g. luciferase).

In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting a DFF-like protein or biologicallyactive portion thereof with a test compound and determining the abilityof the test compound to bind to the DFF-like protein or biologicallyactive portion thereof. Binding of the test compound to the DFF-likeprotein can be determined either directly or indirectly as describedabove. In a preferred embodiment, the assay includes contacting theDFF-like protein or biologically active portion thereof with a knowncompound that binds DFF-like protein to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to preferentially bind to DFF-like proteinor biologically active portion thereof as compared to the knowncompound.

In another embodiment, an assay is a cell-free assay comprisingcontacting DFF-like protein or biologically active portion thereof witha test compound and determining the ability of the test compound tomodulate (e.g., stimulate or inhibit) the activity of the DFF-likeprotein or biologically active portion thereof. Determining the abilityof the test compound to modulate the activity of a DFF-like protein canbe accomplished, for example, by determining the ability of the DFF-likeprotein to bind to a DFF-like target molecule as described above fordetermining direct binding. In an alternative embodiment, determiningthe ability of the test compound to modulate the activity of a DFF-likeprotein can be accomplished by determining the ability of the DFF-likeprotein to further modulate a DFF-like target molecule. For example, thecatalytic/enzymatic activity of the target molecule on an appropriatesubstrate can be determined as previously described.

In yet another embodiment, the cell-free assay comprises contacting theDFF-like protein or biologically active portion thereof with a knowncompound that binds a DFF-like protein to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to preferentially bind to or modulate theactivity of a DFF-like target molecule.

In the above-mentioned assays, it may be desirable to immobilize eithera DFF-like protein or its target molecule to facilitate separation ofcomplexed from uncomplexed forms of one or both of the proteins, as wellas to accommodate automation of the assay. In one embodiment, a fusionprotein can be provided that adds a domain that allows one or both ofthe proteins to be bound to a matrix. For example,glutathione-S-transferase/DFF-like fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione-derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the nonadsorbed targetprotein or DFF-like protein, and the mixture incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads or microtitre plate wellsare washed to remove any unbound components and complex formation ismeasured either directly or indirectly, for example, as described above.Alternatively, the complexes can be dissociated from the matrix, and thelevel of DFF-like binding or activity determined using standardtechniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either DFF-likeprotein or its target molecule can be immobilized utilizing conjugationof biotin and streptavidin. Biotinylated DFF-like molecules or targetmolecules can be prepared from biotin-NHS (N-hydroxy-succinimide) usingtechniques well known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96-well plates (Pierce Chemicals). Alternatively,antibodies reactive with a DFF-like protein or target molecules butwhich do not interfere with binding of the DFF-like protein to itstarget molecule can be derivatized to the wells of the plate, andunbound target or DFF-like protein trapped in the wells by antibodyconjugation. Methods for detecting such complexes, in addition to thosedescribed above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the DFF-likeprotein or target molecule, as well as enzyme-linked assays that rely ondetecting an enzymatic activity associated with the DFF-like protein ortarget molecule.

In another embodiment, modulators of DFF-like expression are identifiedin a method in which a cell is contacted with a candidate compound andthe expression of DFF-like mRNA or protein in the cell is determinedrelative to expression of DFF-like mRNA or protein in a cell in theabsence of the candidate compound. When expression is greater(statistically significantly greater) in the presence of the candidatecompound than in its absence, the candidate compound is identified as astimulator of DFF-like mRNA or protein expression. Alternatively, whenexpression is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound isidentified as an inhibitor of DFF-like mRNA or protein expression. Thelevel of DFF-like mRNA or protein expression in the cells can bedetermined by methods described herein for detecting DFF-like mRNA orprotein.

In yet another aspect of the invention, the DFF-like proteins can beused as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Bio/Techniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and PCT Publication No. WO 94/10300), to identify otherproteins, which bind to or interact with DFF-like protein(“DFF-like-binding proteins” or “DFF-like-bp”) and modulate DFF-likeactivity. Such DFF-like-binding proteins are also likely to be involvedin the propagation of signals by the DFF-like proteins as, for example,upstream or downstream elements of the DFF-like pathway.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(1) map their respective genes on a chromosome; (2) identify anindividual from a minute biological sample (tissue typing); and (3) aidin forensic identification of a biological sample. These applicationsare described in the subsections below.

1. Chromosome Mapping

The isolated complete or partial DFF-like gene sequences of theinvention can be used to map their respective DFF-like genes on achromosome, thereby facilitating the location of gene regions associatedwith genetic disease. Computer analysis of DFF-like sequences can beused to rapidly select PCR primers (preferably 15-25 bp in length) thatdo not span more than one exon in the genomic DNA, thereby simplifyingthe amplification process. These primers can then be used for PCRscreening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the DFF-like sequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow (because they lack a particular enzyme), but in whichhuman cells can, the one human chromosome that contains the geneencoding the needed enzyme will be retained. By using various media,panels of hybrid cell lines can be established. Each cell line in apanel contains either a single human chromosome or a small number ofhuman chromosomes, and a full set of mouse chromosomes, allowing easymapping of individual genes to specific human chromosomes (D'Eustachioet al. (1983) Science 220:919-924). Somatic cell hybrids containing onlyfragments of human chromosomes can also be produced by using humanchromosomes with translocations and deletions.

Other mapping strategies that can similarly be used to map a DFF-likesequence to its chromosome include in situ hybridization (described inFan et al. (1990) Proc. Natl. Acad. Sci. USA 87:6223-27), pre-screeningwith labeled flow-sorted chromosomes, and pre-selection by hybridizationto chromosome specific cDNA libraries. Furthermore, fluorescence in situhybridization (FISH) of a DNA sequence to a metaphase chromosomal spreadcan be used to provide a precise chromosomal location in one step. For areview of this technique, see Verma et al. (1988) Human Chromosomes: AManual of Basic Techniques (Pergamon Press, NY). The FISH technique canbe used with a DNA sequence as short as 500 or 600 bases. However,clones larger than 1,000 bases have a higher likelihood of binding to aunique chromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results in a reasonable amount of time.

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Another strategy to map the chromosomal location of DFF-like genes usesDFF-like polypeptides and fragments and sequences of the presentinvention and antibodies specific thereto. This mapping can be carriedout by specifically detecting the presence of a DFF-like polypeptide inmembers of a panel of somatic cell hybrids between cells of a firstspecies of animal from which the protein originates and cells from asecond species of animal, and then determining which somatic cellhybrid(s) expresses the polypeptide and noting the chromosomes(s) fromthe first species of animal that it contains. For examples of thistechnique, see Pajunen et al. (1988) Cytogenet. Cell. Genet. 47:37-41and Van Keuren et al. (1986) Hum. Genet. 74:34-40. Alternatively, thepresence of a DFF-like polypeptide in the somatic cell hybrids can bedetermined by assaying an activity or property of the polypeptide, forexample, enzymatic activity, as described in Bordelon-Riser et al.(1979) Somatic Cell Genetics 5:597-613 and Owerbach et al. (1978) Proc.Natl. Acad. Sci. USA 75:5640-5644.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship betweengenes and disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, e.g., Egeland et al. (1987) Nature325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the DFF-like gene can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

2. Tissue Typing

The DFF-like sequences of the present invention can also be used toidentify individuals from minute biological samples. The United Statesmilitary, for example, is considering the use of restriction fragmentlength polymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes and probed on a Southern blot to yield unique bandsfor identification. The sequences of the present invention are useful asadditional DNA markers for RFLP (described in U.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique for determining the actual base-by-baseDNA sequence of selected portions of an individual's genome. Thus, theDFF-like sequences of the invention can be used to prepare two PCRprimers from the 5′ and 3′ ends of the sequences. These primers can thenbe used to amplify an individual's DNA and subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The DFF-like sequences of the invention uniquely representportions of the human genome. Allelic variation occurs to some degree inthe coding regions of these sequences, and to a greater degree in thenoncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency of about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. The noncoding sequences of SEQ ID NO:10 cancomfortably provide positive individual identification with a panel ofperhaps 10 to 1,000 primers that each yield a noncoding amplifiedsequence of 100 bases. If a predicted coding sequence, such as that inSEQ ID NO:11, is used, a more appropriate number of primers for positiveindividual identification would be 500 to 2,000.

3. Use of Partial DFF-Like Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. In this manner, PCR technology can be used to amplify DNAsequences taken from very small biological samples such as tissues,e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen foundat a crime scene. The amplified sequence can then be compared to astandard, thereby allowing identification of the origin of thebiological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” that is unique to a particular individual. Asmentioned above, actual base sequence information can be used foridentification as an accurate alternative to patterns formed byrestriction enzyme generated fragments. Sequences targeted to noncodingregions of SEQ ID NO:10 are particularly appropriate for this use asgreater numbers of polymorphisms occur in the noncoding regions, makingit easier to differentiate individuals using this technique. Examples ofpolynucleotide reagents include the DFF-like sequences or portionsthereof, e.g., fragments derived from the noncoding regions of SEQ IDNO:10 having a length of at least 20 or 30 bases.

The DFF-like sequences described herein can further be used to providepolynucleotide reagents, e.g., labeled or labelable probes that can beused in, for example, an in situ hybridization technique, to identify aspecific tissue. This can be very useful in cases where a forensicpathologist is presented with a tissue of unknown origin. Panels of suchDFF-like probes, can be used to identify tissue by species and/or byorgan type.

In a similar fashion, these reagents, e.g., DFF-like primers or probescan be used to screen tissue culture for contamination (i.e., screen forthe presence of a mixture of different types of cells in a culture).

C. Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trails are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. These applications aredescribed in the subsections below.

1. Diagnostic Assays

One aspect of the present invention relates to diagnostic assays fordetecting DFF-like protein and/or nucleic acid expression as well asDFF-like activity, in the context of a biological sample. An exemplarymethod for detecting the presence or absence of DFF-like proteins in abiological sample involves obtaining a biological sample from a testsubject and contacting the biological sample with a compound or an agentcapable of detecting DFF-like protein or nucleic acid (e.g., mRNA,genomic DNA) that encodes DFF-like protein such that the presence ofDFF-like protein is detected in the biological sample. Results obtainedwith a biological sample from the test subject may be compared toresults obtained with a biological sample from a control subject.

“Misexpression or aberrant expression”, as used herein, refers to anon-wild type pattern of gene expression, at the RNA or protein level.It includes: expression at non-wild type levels, i.e., over or underexpression; a pattern of expression that differs from wild type in termsof the time or stage at which the gene is expressed, e.g., increased ordecreased expression (as compared with wild type) at a predetermineddevelopmental period or stage; a pattern of expression that differs fromwild type in terms of decreased expression (as compared with wild type)in a predetermined cell type or tissue type; a pattern of expressionthat differs from wild type in terms of the splicing size, amino acidsequence, post-transitional modification, or biological activity of theexpressed polypeptide; a pattern of expression that differs from wildtype in terms of the effect of an environmental stimulus orextracellular stimulus on expression of the gene, e.g., a pattern ofincreased or decreased expression (as compared with wild type) in thepresence of an increase or decrease in the strength of the stimulus.

A preferred agent for detecting DFF-like mRNA or genomic DNA is alabeled nucleic acid probe capable of hybridizing to DFF-like mRNA orgenomic DNA. The nucleic acid probe can be, for example, a full-lengthDFF-like nucleic acid, such as the nucleic acid of SEQ ID NO:10, or aportion thereof, such as a nucleic acid molecule of at least 15, 30, 50,100, 250, or 500 nucleotides in length and sufficient to specificallyhybridize under stringent conditions to DFF-like mRNA or genomic DNA.Other suitable probes for use in the diagnostic assays of the inventionare described herein.

A preferred agent for detecting DFF-like protein is an antibody capableof binding to DFF-like protein, preferably an antibody with a detectablelabel. Antibodies can be polyclonal, or more preferably, monoclonal. Anintact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can beused. The term “labeled”, with regard to the probe or antibody, isintended to encompass direct labeling of the probe or antibody bycoupling (i.e., physically linking) a detectable substance to the probeor antibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently labeledstreptavidin.

The term “biological sample” is intended to include tissues, cells, andbiological fluids isolated from a subject, as well as tissues, cells,and fluids present within a subject. That is, the detection method ofthe invention can be used to detect DFF-like mRNA, protein, or genomicDNA in a biological sample in vitro as well as in vivo. For example, invitro techniques for detection of DFF-like mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetection of DFF-like protein include enzyme linked immunosorbent assays(ELISAs), Western blots, immunoprecipitations, and immunofluorescence.In vitro techniques for detection of DFF-like genomic DNA includeSouthern hybridizations. Furthermore, in vivo techniques for detectionof DFF-like protein include introducing into a subject a labeledanti-DFF-like antibody. For example, the antibody can be labeled with aradioactive marker whose presence and location in a subject can bedetected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject.

The invention also encompasses kits for detecting the presence ofDFF-like proteins in a biological sample (a test sample). Such kits canbe used to determine if a subject is suffering from or is at increasedrisk of developing a disorder associated with aberrant expression ofDFF-like protein (e.g., a disorder resulting in dysregulated apoptosis).For example, the kit can comprise a labeled compound or agent capable ofdetecting DFF-like protein or mRNA in a biological sample and means fordetermining the amount of a DFF-like protein in the sample (e.g., ananti-DFF-like antibody or an oligonucleotide probe that binds to DNAencoding a DFF-like protein, e.g., SEQ ID NO:10 or 11). Kits can alsoinclude instructions for observing that the tested subject is sufferingfrom or is at risk of developing a disorder associated with aberrantexpression of DFF-like sequences if the amount of DFF-like protein ormRNA is above or below a normal level.

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) that binds to DFF-likeprotein; and, optionally, (2) a second, different antibody that binds toDFF-like protein or the first antibody and is conjugated to a detectableagent. For oligonucleotide-based kits, the kit can comprise, forexample: (1) an oligonucleotide, e.g., a detectably labeledoligonucleotide, that hybridizes to a DFF-like nucleic acid sequence or(2) a pair of primers useful for amplifying a DFF-like nucleic acidmolecule.

The kit can also comprise, e.g., a buffering agent, a preservative, or aprotein stabilizing agent. The kit can also comprise componentsnecessary for detecting the detectable agent (e.g., an enzyme or asubstrate). The kit can also contain a control sample or a series ofcontrol samples that can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container, and all of the various containers are within asingle package along with instructions for observing whether the testedsubject is suffering from or is at risk of developing a disorderassociated with aberrant expression of DFF-like proteins.

2. Other Diagnostic Assays

In another aspect, the invention features, a method of analyzing aplurality of capture probes. The method can be used, e.g., to analyzegene expression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g., anucleic acid or peptide sequence; contacting the array with a DFF-like,preferably purified, nucleic acid, preferably purified, polypeptide,preferably purified, or antibody, and thereby evaluating the pluralityof capture probes. Binding, e.g., in the case of a nucleic acid,hybridization with a capture probe at an address of the plurality, isdetected, e.g., by signal generated from a label attached to theDFF-like nucleic acid, polypeptide, or antibody.

The capture probes can be a set of nucleic acids from a selected sample,e.g., a sample of nucleic acids derived from a control or non-stimulatedtissue or cell.

The method can include contacting the DFF-like nucleic acid,polypeptide, or antibody with a first array having a plurality ofcapture probes and a second array having a different plurality ofcapture probes. The results of each hybridization can be compared, e.g.,to analyze differences in expression between a first and second sample.The first plurality of capture probes can be from a control sample,e.g., a wild type, normal, or non-diseased, non-stimulated, sample,e.g., a biological fluid, tissue, or cell sample. The second pluralityof capture probes can be from an experimental sample, e.g., a mutanttype, at risk, disease-state or disorder-state, or stimulated, sample,e.g., a biological fluid, tissue, or cell sample.

The plurality of capture probes can be a plurality of nucleic acidprobes each of which specifically hybridizes, with an allele ofDFF-like. Such methods can be used to diagnose a subject, e.g., toevaluate risk for a disease or disorder, to evaluate suitability of aselected treatment for a subject, to evaluate whether a subject has adisease or disorder.

The method can be used to detect SNPs, as described above.

In another aspect, the invention features, a method of analyzing aplurality of probes. The method is useful, e.g., for analyzing geneexpression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the pluralityhaving a unique capture probe, e.g., wherein the capture probes are froma cell or subject which express DFF-like or from a cell or subject inwhich a DFF-like mediated response has been elicited, e.g., by contactof the cell with DFF-like nucleic acid or protein, or administration tothe cell or subject DFF-like nucleic acid or protein; contacting thearray with one or more inquiry probe, wherein an inquiry probe can be anucleic acid, polypeptide, or antibody (which is preferably other thanDFF-like nucleic acid, polypeptide, or antibody); providing a twodimensional array having a plurality of addresses, each address of theplurality being positionally distinguishable from each other address ofthe plurality, and each address of the plurality having a unique captureprobe, e.g., wherein the capture probes are from a cell or subject whichdoes not express DFF-like (or does not express as highly as in the caseof the DFF-like positive plurality of capture probes) or from a cell orsubject which in which a DFF-like mediated response has not beenelicited (or has been elicited to a lesser extent than in the firstsample); contacting the array with one or more inquiry probes (which ispreferably other than a DFF-like nucleic acid, polypeptide, orantibody), and thereby evaluating the plurality of capture probes.Binding, e.g., in the case of a nucleic acid, hybridization with acapture probe at an address of the plurality, is detected, e.g., bysignal generated from a label attached to the nucleic acid, polypeptide,or antibody.

In another aspect, the invention features, a method of analyzingDFF-like, e.g., analyzing structure, function, or relatedness to othernucleic acid or amino acid sequences. The method includes: providing aDFF-like nucleic acid or amino acid sequence, comparing the DFF-likesequence with one or more preferably a plurality of sequences from acollection of sequences, e.g., a nucleic acid or protein sequencedatabase; to thereby analyze DFF-like.

The method can include evaluating the sequence identity between aDFF-like sequence and a database sequence. The method can be performedby accessing the database at a second site, e.g., over the internet.

In another aspect, the invention features, a set of oligonucleotides,useful, e.g., for identifying SNP's, or identifying specific alleles ofDFF-like. The set includes a plurality of oligonucleotides, each ofwhich has a different nucleotide at an interrogation position, e.g., anSNP or the site of a mutation. In a preferred embodiment, theoligonucleotides of the plurality identical in sequence with one another(except for differences in length). The oligonucleotides can be providedwith differential labels, such that an oligonucleotides which hybridizesto one allele provides a signal that is distinguishable from anoligonucleotides which hybridizes to a second allele.

3. Prognostic Assays

The methods described herein can furthermore be utilized as diagnosticor prognostic assays to identify subjects having or at risk ofdeveloping a disease or disorder associated with DFF-like protein,DFF-like nucleic acid expression, or DFF-like activity. Prognosticassays can be used for prognostic or predictive purposes to therebyprophylactically treat an individual prior to the onset of a disordercharacterized by or associated with DFF-like protein, DFF-like nucleicacid expression, or DFF-like activity.

Thus, the present invention provides a method in which a test sample isobtained from a subject, and DFF-like protein or nucleic acid (e.g.,mRNA, genomic DNA) is detected, wherein the presence of DFF-like proteinor nucleic acid is diagnostic for a subject having or at risk ofdeveloping a disease or disorder associated with aberrant DFF-likeexpression or activity. As used herein, a “test sample” refers to abiological sample obtained from a subject of interest. For example, atest sample can be a biological fluid (e.g., serum), cell sample, ortissue.

Furthermore, using the prognostic assays described herein, the presentinvention provides methods for determining whether a subject can beadministered a specific agent (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, nucleic acid, small molecule, or otherdrug candidate) or class of agents (e.g., agents of a type that decreaseDFF-like activity) to effectively treat a disease or disorder associatedwith aberrant DFF-like expression or activity. In this manner, a testsample is obtained and DFF-like protein or nucleic acid is detected. Thepresence of DFF-like protein or nucleic acid is diagnostic for a subjectthat can be administered the agent to treat a disorder associated withaberrant DFF-like expression or activity.

The methods of the invention can also be used to detect genetic lesionsor mutations in a DFF-like gene, thereby determining if a subject withthe lesioned gene is at risk for a disorder characterized bydysregulated apoptosis. In preferred embodiments, the methods includedetecting, in a sample of cells from the subject, the presence orabsence of a genetic lesion or mutation characterized by at least one ofan alteration affecting the integrity of a gene encoding aDFF-like-protein, or the misexpression of the DFF-like gene. Forexample, such genetic lesions or mutations can be detected byascertaining the existence of at least one of: (1) a deletion of one ormore nucleotides from a DFF-like gene; (2) an addition of one or morenucleotides to a DFF-like gene; (3) a substitution of one or morenucleotides of a DFF-like gene; (4) a chromosomal rearrangement of aDFF-like gene; (5) an alteration in the level of a messenger RNAtranscript of a DFF-like gene; (6) an aberrant modification of aDFF-like gene, such as of the methylation pattern of the genomic DNA;(7) the presence of a non-wild-type splicing pattern of a messenger RNAtranscript of a DFF-like gene; (8) a non-wild-type level of aDFF-like-protein; (9) an allelic loss of a DFF-like gene; and (10) aninappropriate post-translational modification of a DFF-like-protein. Asdescribed herein, there are a large number of assay techniques known inthe art that can be used for detecting lesions in a DFF-like gene. Anycell type or tissue in which DFF-like proteins are expressed may beutilized in the prognostic assays described herein.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the DFF-like-gene(see, e.g., Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). It isanticipated that PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

Alternative amplification methods include self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a DFF-like gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns of isolated test sample and control DNA digested with one ormore restriction endonucleases. Moreover, the use of sequence specificribozymes (see, e.g., U.S. Pat. No. 5,498,531) can be used to score forthe presence of specific mutations by development or loss of a ribozymecleavage site.

In other embodiments, genetic mutations in a DFF-like molecule can beidentified by hybridizing a sample and control nucleic acids, e.g., DNAor RNA, to high density arrays containing hundreds or thousands ofoligonucleotides probes (Cronin et al. (1996) Human Mutation 7:244-255;Kozal et al. (1996) Nature Medicine 2:753-759). In yet anotherembodiment, any of a variety of sequencing reactions known in the artcan be used to directly sequence the DFF-like gene and detect mutationsby comparing the sequence of the sample DF-like gene with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxim andGilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977)Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any ofa variety of automated sequencing procedures can be utilized whenperforming the diagnostic assays ((1995) Bio/Techniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT PublicationNo. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36: 127-162; andGriffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

Other methods for detecting mutations in the DFF-like gene includemethods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al.(1985) Science 230:1242). See, also Cotton et al. (1988) Proc. Natl.Acad. Sci. USA 85:4397; Saleeba et al. (1992) Methods Enzymol.217:286-295. In a preferred embodiment, the control DNA or RNA can belabeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more “DNA mismatch repair” enzymes that recognize mismatched basepairs in double-stranded DNA in defined systems for detecting andmapping point mutations in DFF-like cDNAs obtained from samples ofcells. See, e.g., Hsu et al. (1994) Carcinogenesis 15:1657-1662.According to an exemplary embodiment, a probe based on a DFF-likesequence, e.g., a wild-type DFF-like sequence, is hybridized to a cDNAor other DNA product from a test cell(s). The duplex is treated with aDNA mismatch repair enzyme, and the cleavage products, if any, can bedetected from electrophoresis protocols or the like. See, e.g., U.S.Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in DFF-like genes. For example, single-strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild-type nucleic acids(Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766; see also Cotton(1993) Mutat. Res. 285:125-144; Hayashi (1992) Genet. Anal. Tech. Appl.9:73-79). The sensitivity of the assay may be enhanced by using RNA(rather than DNA), in which the secondary structure is more sensitive toa change in sequence. In a preferred embodiment, the subject methodutilizes heteroduplex analysis to separate double-stranded heteroduplexmolecules on the basis of changes in electrophoretic mobility (Keen etal. (1991) Trends Genet. 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditions thatpermit hybridization only if a perfect match is found (Saiki et al.(1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci. USA86:6230). Such allele-specific oligonucleotides are hybridized toPCR-amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele-specific amplification technology, which dependson selective PCR amplification, may be used in conjunction with theinstant invention. Oligonucleotides used as primers for specificamplification may carry the mutation of interest in the center of themolecule so that amplification depends on differential hybridization(Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme3′ end of one primer where, under appropriate conditions, mismatch canprevent or reduce polymerase extension (Prossner (1993) Tibtech 11:238).In addition, it may be desirable to introduce a novel restriction sitein the region of the mutation to create cleavage-based detection(Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated thatin certain embodiments amplification may also be performed using Taqligase for amplification (Barany (1991) Proc. Natl. Acad. Sci. USA88:189). In such cases, ligation will occur only if there is a perfectmatch at the 3′ end of the 5′ sequence making it possible to detect thepresence of a known mutation at a specific site by looking for thepresence or absence of amplification.

The methods described herein may be performed, for example, by utilizingprepackaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnosed patients exhibiting symptoms orfamily history of a disease or illness involving a DFF-like gene.

4. Pharmacogenomics

Agents, or modulators that have a stimulatory or inhibitory effect onDFF-like activity (e.g., DFF-like gene expression) as identified by ascreening assay described herein, can be administered to individuals totreat (prophylactically or therapeutically) disorders associated withaberrant DFF-like activity as well as to modulate the phenotype ofdysregulated apoptosis. In conjunction with such treatment, thepharmacogenomics (i.e., the study of the relationship between anindividual's genotype and that individual's response to a foreigncompound or drug) of the individual may be considered. Differences inmetabolism of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, the pharmacogenomics of theindividual permits the selection of effective agents (e.g., drugs) forprophylactic or therapeutic treatments based on a consideration of theindividual's genotype. Such pharmacogenomics can further be used todetermine appropriate dosages and therapeutic regimens. Accordingly, theactivity of DFF-like protein, expression of DFF-like nucleic acid, ormutation content of DFF-like genes in an individual can be determined tothereby select appropriate agent(s) for therapeutic or prophylactictreatment of the individual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Linder (1997) Clin. Chem.43(2):254-266. In general, two types of pharmacogenetic conditions canbe differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body are referred to as “altered drugaction.” Genetic conditions transmitted as single factors altering theway the body acts on drugs are referred to as “altered drug metabolism”.These pharmacogenetic conditions can occur either as rare defects or aspolymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency(G6PD) is a common inherited enzymopathy in which the main clinicalcomplication is haemolysis after ingestion of oxidant drugs(antimalarials, sulfonamides, analgesics, nitrofurans) and consumptionof fava beans.

Differences in metabolism of therapeutics can lead to severe toxicity ortherapeutic failure by altering the relation between dose and bloodconcentration of the pharmacologically active drug. Thus, a physician orclinician may consider applying knowledge obtained in relevantpharmacogenomics studies in determining whether to administer a DFF-likemolecule or DFF-like modulator as well as tailoring the dosage and/ortherapeutic regimen of treatment with a DFF-like molecule or DFF-likemodulator.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drug's target is known (e.g., aDFF-like protein of the present invention), all common variants of thatgene can be fairly easily identified in the population and it can bedetermined if having one version of the gene versus another isassociated with a particular drug response.

Alternatively, a method termed the “gene expression profiling”, can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., a DFF-likemolecule or DFF-like modulator of the present invention) can give anindication whether gene pathways related to toxicity have been turnedon.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment of an individual.This knowledge, when applied to dosing or drug selection, can avoidadverse reactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a DFF-like moleculeor DFF-like modulator, such as a modulator identified by one of theexemplary screening assays described herein.

The present invention further provides methods for identifying newagents, or combinations, that are based on identifying agents thatmodulate the activity of one or more of the gene products encoded by oneor more of the DFF-like genes of the present invention, wherein theseproducts may be associated with resistance of the cells to a therapeuticagent. Specifically, the activity of the proteins encoded by theDFF-like genes of the present invention can be used as a basis foridentifying agents for overcoming agent resistance. By blocking theactivity of one or more of the resistance proteins, target cells willbecome sensitive to treatment with an agent that the unmodified targetcells were resistant to.

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a DFF-like protein can be applied in clinical trials. Forexample, the effectiveness of an agent determined by a screening assayas described herein to increase DFF-like gene expression, proteinlevels, or upregulate DFF-like activity, can be monitored in clinicaltrials of subjects exhibiting decreased DFF-like gene expression,protein levels, or downregulated DFF-like activity. Alternatively, theeffectiveness of an agent determined by a screening assay to decreaseDFF-like gene expression, protein levels, or downregulate DFF-likeactivity, can be monitored in clinical trials of subjects exhibitingincreased DFF-like gene expression, protein levels, or upregulatedDFF-like activity. In such clinical trials, the expression or activityof a DFF-like gene, and preferably, other genes that have beenimplicated in, for example, a DFF-like-associated disorder can be usedas a “read out” or markers of the phenotype of a particular cell.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, a PM will show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the activity of DFF-like protein, expression of DFF-like nucleicacid, or mutation content of DFF-like genes in an individual can bedetermined to thereby select appropriate agent(s) for therapeutic orprophylactic treatment of the individual. In addition, pharmacogeneticstudies can be used to apply genotyping of polymorphic alleles encodingdrug-metabolizing enzymes to the identification of an individual's drugresponsiveness phenotype. This knowledge, when applied to dosing or drugselection, can avoid adverse reactions or therapeutic failure and thusenhance therapeutic or prophylactic efficiency when treating a subjectwith a DFF-like modulator, such as a modulator identified by one of theexemplary screening assays described herein.

5. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of DFF-like genes (e.g., the ability to modulateapoptotic events) can be applied not only in basic drug screening butalso in clinical trials. For example, the effectiveness of an agent, asdetermined by a screening assay as described herein, to increase ordecrease DFF-like gene expression, protein levels, or protein activity,can be monitored in clinical trials of subjects exhibiting decreased orincreased DFF-like gene expression, protein levels, or protein activity.In such clinical trials, DFF-like expression or activity and preferablythat of other genes that have been implicated in for example, a disorderresulting in dysregulated apoptosis, can be used as a marker of anapoptotic events of a particular cell.

For example, and not by way of limitation, genes that are modulated incells by treatment with an agent (e.g., compound, drug, or smallmolecule) that modulates DFF-like activity (e.g., as identified in ascreening assay described herein) can be identified. Thus, to study theeffect of agents on cellular disorders resulting from dysregulatedapoptosis, for example, in a clinical trial, cells can be isolated andRNA prepared and analyzed for the levels of expression of DFF-like genesand other genes implicated in the disorder. The levels of geneexpression (i.e., a gene expression pattern) can be quantified byNorthern blot analysis or RT-PCR, as described herein, or alternativelyby measuring the amount of protein produced, by one of the methods asdescribed herein, or by measuring the levels of activity of DFF-likegenes or other genes. In this way, the gene expression pattern can serveas a marker, indicative of the physiological response of the cells tothe agent. Accordingly, this response state may be determined before,and at various points during, treatment of the individual with theagent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (1) obtaininga preadministration sample from a subject prior to administration of theagent; (2) detecting the level of expression of a DFF-like protein,mRNA, or genomic DNA in the preadministration sample; (3) obtaining oneor more postadministration samples from the subject; (4) detecting thelevel of expression or activity of the DFF-like protein, mRNA, orgenomic DNA in the postadministration samples; (5) comparing the levelof expression or activity of the DFF-like protein, mRNA, or genomic DNAin the preadministration sample with the DFF-like protein, mRNA, orgenomic DNA in the postadministration sample or samples; and (vi)altering the administration of the agent to the subject accordingly tobring about the desired effect, i.e., for example, an increase or adecrease in the expression or activity of a DFF-like protein.

C. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant DFF-like expression oractivity. “Subject”, as used herein, can refer to a mammal, e.g., ahuman, or to an experimental or animal or disease model. The subject canalso be a non-human animal, e.g., a horse, cow, goat, or other domesticanimal. Additionally, the compositions of the invention find use in thetreatment of disorders described herein. Thus, therapies for disordersassociated with a DFF-like molecule are encompassed herein. “Treatment”is herein defined as the application or administration of a therapeuticagent to a patient, or application or administration of a therapeuticagent to an isolated tissue or cell line from a patient, who has adisease, a symptom of disease or a predisposition toward a disease, withthe purpose to cure, heal, alleviate, relieve, alter, remedy,ameliorate, improve or affect the disease, the symptoms of disease orthe predisposition toward disease. A “therapeutic agent” includes, butis not limited to, small molecules, peptides, antibodies, ribozymes andantisense oligonucleotides.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject a disease or condition associated with an aberrant DFF-likeexpression or activity by administering to the subject an agent thatmodulates DFF-like expression or at least one DFF-like gene activity.Subjects at risk for a disease that is caused, or contributed to, byaberrant DFF-like expression or activity can be identified by, forexample, any or a combination of diagnostic or prognostic assays asdescribed herein. Administration of a prophylactic agent can occur priorto the manifestation of symptoms characteristic of the DFF-likeaberrancy, such that a disease or disorder is prevented or,alternatively, delayed in its progression. Depending on the type ofDFF-like aberrancy, for example, a DFF-like agonist or DFF-likeantagonist agent can be used for treating the subject. The appropriateagent can be determined based on screening assays described herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulatingDFF-like expression or activity for therapeutic purposes. The modulatorymethod of the invention involves contacting a cell with an agent thatmodulates one or more of the activities of DFF-like protein activityassociated with the cell. An agent that modulates DFF-like proteinactivity can be an agent as described herein, such as a nucleic acid ora protein, a naturally-occurring cognate ligand of a DFF-like protein, apeptide, a DFF-like peptidomimetic, or other small molecule. In oneembodiment, the agent stimulates one or more of the biologicalactivities of DFF-like protein. Examples of such stimulatory agentsinclude active DFF-like protein and a nucleic acid molecule encoding aDFF-like protein that has been introduced into the cell. In anotherembodiment, the agent inhibits one or more of the biological activitiesof DFF-like protein. Examples of such inhibitory agents includeantisense DFF-like nucleic acid molecules and anti-DFF-like antibodies.

These modulatory methods can be performed in vitro (e.g., by culturingthe cell with the agent) or, alternatively, in vivo (e.g, byadministering the agent to a subject). As such, the present inventionprovides methods of treating an individual afflicted with a disease ordisorder characterized by aberrant expression or activity of a DFF-likeprotein or nucleic acid molecule. In one embodiment, the method involvesadministering an agent (e.g., an agent identified by a screening assaydescribed herein), or a combination of agents, that modulates (e.g.,upregulates or downregulates) DFF-like expression or activity. Inanother embodiment, the method involves administering a DFF-like proteinor nucleic acid molecule as therapy to compensate for reduced oraberrant DFF-like expression or activity.

Stimulation of DFF-like activity is desirable in situations in which aDFF-like protein is abnormally downregulated and/or in which increasedDFF-like activity is likely to have a beneficial effect. Conversely,inhibition of DFF-like activity is desirable in situations in whichDFF-like activity is abnormally upregulated and/or in which decreasedDFF-like activity is likely to have a beneficial effect.

This invention is further illustrated by the following examples, whichshould not be construed as limiting.

Example 1 Identification and Characterization of Human DFF-Like cDNAs

The human DFF-like sequence (SEQ ID NO:10), which is approximately 1284nucleotides long including untranslated regions, contains a predictedmethionine-initiated coding sequence of about 169 nucleotides(nucleotides 169-828 of SEQ ID NO:10). The coding sequence encodes a 219amino acid protein (SEQ ID NO:11).

A search of the nucleotide and protein databases revealed that 5698encodes a precursor polypeptide that shares similarity with several CIDEproteins. An alignment of the protein sequences having highestsimilarity to the 5698 precursor polypeptide is shown in FIG. 19. Thealignment was generated using the Clustal method with PAM250 residueweight table and sequence identities were determined by FASTA (Pearsonand Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448).

Example 2 Tissue Distribution of a DFF-Like mRNA

Northern blot hybridizations with various RNA samples can be performedunder standard conditions and washed under stringent conditions, i.e.,0.2×SSC at 65° C. A DNA probe corresponding to all or a portion of theDFF-like cDNA (SEQ ID NO:10 or 3) can be used. The DNA was radioactivelylabeled with ³²P-dCTP using the Prime-It Kit (Stratagene, La Jolla,Calif.) according to the instructions of the supplier. Filterscontaining mRNA from mouse hematopoietic and endocrine tissues, andcancer cell lines (Clontech, Palo Alto, Calif.) can be probed inExpressHyb hybridization solution (Clontech) and washed at highstringency according to manufacturer's recommendations.

TaqMan analysis of 5698 revealed expression in a number of tissues. See,for example, FIGS. 20A-B. A high level of expression was seen in liver,diseased and normal heart ventricle, normal kidney, kidney HT, atrium ofnormal heart, diseased heart ventricle, fetal heart, and skeletalmuscle. The source number associated with each of these tissue types isprovided below in Table 1. TABLE 1 Source # CV Organ MPI 1097 H HeartNormal Atrium pit 277 H Heart Normal Atrium pit 272 H Heart NormalVentricle tlo⁻1 H Heart Normal Ventricle pit 278 H Heart NormalVentricle pit 204 H Heart Normal Ventricle pit 205 H Heart NormalVentricle eli 5 H Heart Diseased Ventricle pit 16 H Heart DiseasedVentricle mpi 613 H Heart Diseased Ventricle bwh 4 H Fetal Heart ndr 171H Kidney normal ndr 179 H Kidney normal pit 289 H Kidney normal pit 351H Kidney normal pit 353 H Kidney normal ndr 233 H Kidney HT ndr 224 HKidney HT ndr 248 H Kidney HT ndr 252 H Kidney HT CHT 1176 H Kidney HTmpi 570 H Skeletal Muscle mpi 602 H Skeletal Muscle pit 284 H SkeletalMuscle mpi 145 H Liver mpi 155 H Liver mpi 146 H Liver MPI 90 M HeartNormal Atrium MPI 92 M Heart Normal Atrium MPI 96 M Heart NormalVentricle MPI 538 M Heart Normal Ventricle

Example 3 Recombinant Expression of DFF-Like Sequences in BacterialCells

In this example, the DFF-like sequence is expressed as a recombinantglutathione-S-transferase (GST) fusion polypeptide in E. coli and thefusion polypeptide is isolated and characterized. Specifically, theDFF-like sequence is fused to GST and this fusion polypeptide isexpressed in E. coli, e.g., strain PEB199. Expression of theGST-DFF-like fusion protein in PEB199 is induced with IPTG. Therecombinant fusion polypeptide is purified from crude bacterial lysatesof the induced PEB199 strain by affinity chromatography on glutathionebeads. Using polyacrylamide gel electrophoretic analysis of thepolypeptide purified from the bacterial lysates, the molecular weight ofthe resultant fusion polypeptide is determined.

Example 4 Expression of Recombinant DFF-Like Protein in COS Cells

To express the DFF-like gene in COS cells, the pcDNA/Amp vector byInvitrogen Corporation (San Diego, Calif.) is used. This vector containsan SV40 origin of replication, an ampicillin resistance gene, an E. colireplication origin, a CMV promoter followed by a polylinker region, andan SV40 intron and polyadenylation site. A DNA fragment encoding theentire DFF-like protein and an HA tag (Wilson et al. (1984) Cell 37:767)or a FLAG tag fused in-frame to its 3′ end of the fragment is clonedinto the polylinker region of the vector, thereby placing the expressionof the recombinant protein under the control of the CMV promoter.

To construct the plasmid, the DFF-like DNA sequence is amplified by PCRusing two primers. The 5′ primer contains the restriction site ofinterest followed by approximately twenty nucleotides of the DFF-likecoding sequence starting from the initiation codon; the 3′ end sequencecontains complementary sequences to the other restriction site ofinterest, a translation stop codon, the HA tag or FLAG tag and the last20 nucleotides of the DFF-like coding sequence. The PCR amplifiedfragment and the pCDNA/Amp vector are digested with the appropriaterestriction enzymes and the vector is dephosphorylated using the CIAPenzyme (New England Biolabs, Beverly, Mass.). Preferably the tworestriction sites chosen are different so that the DFF-like gene isinserted in the correct orientation. The ligation mixture is transformedinto E. coli cells (strains HB101, DH5α, SURE, available from StratageneCloning Systems, La Jolla, Calif., can be used), the transformed cultureis plated on ampicillin media plates, and resistant colonies areselected. Plasmid DNA is isolated from transformants and examined byrestriction analysis for the presence of the correct fragment.

COS cells are subsequently transfected with the DFF-like-pcDNA/Ampplasmid DNA using the calcium phosphate or calcium chlorideco-precipitation methods, DEAE-dextran-mediated transfection,lipofection, or electroporation. Other suitable methods for transfectinghost cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989. The expression of the DFF-like polypeptide is detected byradiolabelling (³⁵S-methionine or ³⁵S-cysteine available from NEN,Boston, Mass., can be used) and immunoprecipitation (Harlow, E. andLane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1988) using an HA specific monoclonalantibody. Briefly, the cells are labeled for 8 hours with ³⁵S-methionine(or ³⁵S-cysteine). The culture media are then collected and the cellsare lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1%SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culturemedia are precipitated with an HA specific monoclonal antibody.Precipitated polypeptides are then analyzed by SDS-PAGE.

Alternatively, DNA containing the DFF-like coding sequence is cloneddirectly into the polylinker of the pCDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of theDFF-like polypeptide is detected by radiolabelling andimmunoprecipitation using a DFF-like specific monoclonal antibody.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

Chapter 4 32621, Novel Human Phospholipid Scramblase-Like Molecules andUses Thereof BACKGROUND OF THE INVENTION

Phospholipid asymmetry is a well-known characteristic of mammalianplasma membranes. The outer leaflet of the lipid bilayer is rich incholine-phospholipids, whereas aminophospholipids are preferentially inthe inner leaflet (Bevers, E. M. et al., (1998) Lupus Suppl. 2:S126-S131). The plasma membrane phospholipids of erythrocytes (RBC),platelets, and vascular endothelium are normally asymmetricallydistributed. Phosphatidylserine (PS) and phosphatidylethanolamine (PE)reside almost exclusively in the inner leaflet, and thephosphatidylcholine (PC) and sphyingomyelin are enriched in the outerleaflet. This asymmetric distribution of PL is maintained by anaminophospholipid translocase (APLT) which is a Mg⁺²-dependent ATPasethat transports PS and PE, but not PC, from the outer to the innerplasma membrane leaflet (Stout, J. G., et al. (1997) J. Clin. Invest.99(9): 2232-2238).

The APLT activity has now been identified in numerous cell types,including platelets, lymphocytes, fibroblasts, and synaptosomes,suggesting that the asymmetry might be a general property of all cells(Woon, L. A., et al., (1999) Cell Calcium 25(4):313-320).

When PS and PE become exposed on the outer membrane leaflet by variousmechanisms of cell activation, the Mg⁺²-ATPase activity of APLT restoresphospholipid asymmetry by transporting these lipids to the inner bilayerleaflet. A number of physiological and pathophysiological conditions mayresult in the disruption of the normal phospholipid asymmetry of theplasma membrane leading to the exposure of PS on the surface of cells.Exposure of PS creates a procoagulant surface on platelets,erythrocytes, and vascular endothelial cells. Also, there is evidencewhich indicates that clotting, cellular adhesion, fusion andphagocytosis of senescent or apoptotic cells are dependent on PSexposure (Woon et al. (1999) Cell Calcium 25(4):313-320).

A second mechanism which causes phospholipid redistribution in theplasma membrane has been linked to a phospholipid (PL) scramblase. Thescramblase is an integral membrane protein that can mimic the action ofCa⁺² at the endothelial surface of the erythrocyte membrane (Zhao et al.(1998) J. Biol. Chem. 273(12):6603-6606). Zhao et al. demonstrated thatthe propensity for PS to become exposed at the cell surface can bemanipulated by altering the level of expression of PL scramblase throughplasmid transfection. Zhao et al. posit that the transfection of cellswith PL scramblase cDNA promotes movement of PS to the cell surface andsuggests that this protein is involved in the normal redistribution ofplasma membrane phospholipids in activated, injured, and apoptoticcells.

Phospholipid (PL) scramblase is a plasma membrane protein that mediatesaccelerated transbilayer migration of PLs, upon binding of Ca⁻²,facilitating rapid mobilization of phosphatidylserine to the cellsurface upon elevation of internal calcium. (Stout, J. G. et al., (1998)Biochemistry 37:14860-14866). An increase in intracellular calcium dueto cell activation, injury, or apoptosis causes rapid bidirectionalmovement of plasma membrane PL between leaflets. PL scramblase isresponsible for this two-way movement of PL between the membraneleaflets, resulting in exposure of PS and PE at the cell surface(Kasukabe, T. et al., (1998) Biochem. and Biophys. Res. Commun. 249:449-455). The PL scramblase can be assayed using methods as described byZhou (Zhou, Q. et al., (1998) Biochemistry 37: 2356-2360).

One important clinical disorder which may be linked to defective PLscramblase is Scott syndrome. Scott syndrome is a congenital bleedingdisorder related to defective expression of membrane coagulant activity.Circulating blood cells show decreased cell surface exposure ofphosphatidylserine (PS) at elevated cystolic Ca⁺² indicating a defect ordeficiency in PL scramblase (Stout, J. G. et al., (1997) J. Clin.Invest. 99(9):2232-2238). Scott syndrome is an extremely rare bleedingdisorder associated with a defect of the outward transmembrane migrationof pro-coagulant phospholipids at the surface of stimulated platelets orderived-microparticles. Scott syndrome is transmitted as an autosomalrecessive trait as demonstrated in a familial study (Toti, F. et al.,(1996) Blood 87:1409-1415).

Recently, the molecular cloning of murine and human PL scramblases hasbeen reported. Zhou et al. reported the cDNA cloning of a 37-kDa humanplasma membrane phospholipid scramblase from human erythrocytes (Zhou,Q. et al., (1997) J. Biol. Chem. 272(29): 18240-18244). Antibody to thescramblase indicated an approximately 10-fold higher abundance of the PLscramblase in platelets as compared to erythrocytes. The work of Zhou etal. indicated that PL scramblase mRNA is found in a variety ofhematalogic and nonhematologic cells and tissues. The resulting exposureof PS at the cell surface is thought to play a key role in thereticuloendothelial system, in addition to activation of both the plasmacomplement and coagulation systems.

More recently, the cDNA cloning of a human plasma membrane PL scramblase(MmTRA1b) from human monocytic U937 cells and the chromosome mapping ofthe gene was reported (Kaskube, T. et al., (1998) Biochem. and Biophys.Res. Comm. 249: 449-455). The MmTRA1b gene is the human homologue of thepreviously cloned mouse leukemogenesis-associated gene (MmTRA1a). Themouse MmTRA1a is the truncated form of mouse MmTRA1b. The human MmTRA1bcDNA predicted a 318 amino acid protein with a molecular weight of35,047 Da.

The human MmTRA1b protein sequence shared a 78% amino acid identity withthe mouse counterpart (328 amino acids). The human MmTRA1b gene wasmapped to chromosome 3q23. Expression of the human homologue wasincreased during differentiation of U937 cells by most typicaldifferentiation inducers. Also, the predicted amino acid sequenceanalysis of the human MmTRA1b cDNA revealed perfect identity with humanplasma membrane phospholipid scramblase that is required fortransbilayer movement of membrane phospholipids (Kaskukabe, T. et al.,(1998) Biochem. and Biophys. Res. Comm. 249: 449-455).

According to homology searches against EMBL/Genbank/DDBJ data basesthere are at least three homologous C. elegans genes which are moreclosely related with the mouse and human MmTRA1b than previouslyreported, as detailed by Kasukabe et al. Therefore, there appear to beat least two mouse genes (MmTRA1b and PL scramblase as reported by Zhouet al. and five C. elegans genes which constitute a whole new family ofPL flip/flop genes.

The human phospholipid scramblase gene herein described may play animportant role in erythrocyte, platelet, lymphocyte and endotheliumphysiology and function. It may play a particularly important role inthe treatment and diagnosis of bleeding disorders such as Scott syndromeand other hematologic disease conditions, including but not limited tolymphocytic disorders, plasma cell dyscrasias, hemolytic anemias,autoimmune neutropenias, immune thrombocytopenias, lymphocyticleukemias, leukopenia, lymphomas, red cell disorders, plateletdisorders, and coagulation disorders.

SUMMARY OF THE INVENTION

Isolated nucleic acid molecules corresponding to human phospholipidscramblase-like nucleic acid sequences are provided. Additionally, aminoacid sequences corresponding to the polynucleotides are encompassed. Inparticular, the present invention provides for isolated nucleic acidmolecules comprising nucleotide sequences encoding the amino acidsequences shown in SEQ ID NO:17. Further provided are human phospholipidscramblase-like polypeptides having an amino acid sequence encoded by anucleic acid molecule described herein.

The present invention also provides vectors and host cells forrecombinant expression of the nucleic acid molecules described herein,as well as methods of making such vectors and host cells and for usingthem for production of the polypeptides or peptides of the invention byrecombinant techniques.

The human phospholipid scramblase-like molecules of the presentinvention are useful for modulating the immune, hematopoietic, and bloodclotting systems. The molecules are useful for the diagnosis andtreatment of disorders relevant but not limited to erythrocytes,platelets, endothelial and other cells and tissues known to exposeplasma membrane phospholipid in response to elevated cystolic Ca⁺².Additionally, the molecules of the invention are useful as modulatingagents in a variety of cellular processes where the transbilayermovement of phospholipids in the plasma membrane is important for propercellular function and homeostasis.

Accordingly, in one aspect, this invention provides isolated nucleicacid molecules encoding human phospholipid scramblase-like proteins orbiologically active portions thereof, as well as nucleic acid fragmentssuitable as primers or hybridization probes for the detection of humanphospholipid scramblase-like encoding nucleic acids.

Another aspect of this invention features isolated or recombinant humanphospholipid scramblase-like proteins and polypeptides. Preferred humanphospholipid scramblase-like proteins and polypeptides possess at leastone biological activity possessed by naturally occurring humanphospholipid scramblase-like proteins.

Variant nucleic acid molecules and polypeptides substantially homologousto the nucleotide and amino acid sequences set forth in the sequencelistings are encompassed by the present invention.

Antibodies and antibody fragments that selectively bind the humanphospholipid scramblase-like polypeptides and fragments are provided.Such antibodies are useful in detecting the human phospholipidscramblase-like polypeptides.

In another aspect, the present invention provides a method for detectingthe presence of human phospholipid scramblase-like activity orexpression in a biological sample by contacting the biological samplewith an agent capable of detecting an indicator of human phospholipidscramblase-like activity such that the presence of human phospholipidscramblase-like activity is detected in the biological sample.

In yet another aspect, the invention provides a method for modulatinghuman phospholipid scramblase-like activity comprising contacting a cellwith an agent that modulates (inhibits or stimulates) human phospholipidscramblase-like activity or expression such that human phospholipidscramblase-like activity or expression in the cell is modulated. In oneembodiment, the agent is an antibody that specifically binds to humanphospholipid scramblase-like protein. In another embodiment, the agentmodulates expression of human phospholipid scramblase-like protein bymodulating transcription of a human phospholipid scramblase-like gene,splicing of a human phospholipid scramblase-like mRNA, or translation ofa human phospholipid scramblase-like mRNA. In yet another embodiment,the agent is a nucleic acid molecule having a nucleotide sequence thatis antisense to the coding strand of the human phospholipidscramblase-like mRNA or the human phospholipid scramblase-like gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant humanphospholipid scramblase-like protein activity or nucleic acid expressionby administering an agent that is a human phospholipid scramblase-likemodulator to the subject. In one embodiment, the human phospholipidscramblase-like modulator is a human phospholipid scramblase-likeprotein. In another embodiment, the human phospholipid scramblase-likemodulator is a human phospholipid scramblase-like nucleic acid molecule.In other embodiments, the human phospholipid scramblase-like modulatoris a peptide, peptidomimetic, or other small molecule.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic lesion or mutation characterized byat least one of the following: (1) aberrant modification or mutation ofa gene encoding a human phospholipid scramblase-like protein; (2)misregulation of a gene encoding a human phospholipid scramblase-likeprotein; and (3) aberrant post-translational modification of a humanphospholipid scramblase-like protein, wherein a wild-type form of thegene encodes a protein with a human phospholipid scramblase-likeactivity.

In another aspect, the invention provides a method for identifying acompound that binds to or modulates the activity of a human phospholipidscramblase-like protein. In general, such methods entail measuring abiological activity of a human phospholipid scramblase-like protein inthe presence and absence of a test compound and identifying thosecompounds that alter the activity of the human phospholipidscramblase-like protein.

The invention also features methods for identifying a compound thatmodulates the expression of human phospholipid scramblase-like genes bymeasuring the expression of the human phospholipid scramblase-likesequences in the presence and absence of the compound.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the invention are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

The present invention provides phospholipid scramblase-like molecules.By “phospholipid scramblase-like” is intended a novel human sequencereferred to as 32621, and variants and fragments thereof. Thesefull-length gene sequences or fragments thereof are referred to as“phospholipid scramblase-like” sequences, indicating they share sequencesimilarity with phospholipid scramblase genes. Isolated nucleic acidmolecules comprising nucleotide sequences encoding the 32621 polypeptidewhose amino acid sequence is given in SEQ ID NO:17, or a variant orfragment thereof, are provided. A nucleotide sequence encoding the 32621polypeptide is set forth in SEQ ID NO:16 and SEQ ID NO:18.

The disclosed invention relates to methods and compositions for themodulation, diagnosis, and treatment of immune, hematopoietic, platelet,and blood coagulation disorders. Such immune disorders include, but notlimited to, lymphocytic disorders, plasma cell dyscrasias, hemolyticanemias, autoimmune neutropenias, immune thrombocytopenias, lymphocyticleukemias, leukopenia, and lymphomas. The hematopoietic disordersinclude, but are not limited to, all bone marrow and red blood celldisorders. The blood coagulation disorders include, but are not limitedto, hemophilia and Von Willebrand's disease. Platelet disorders include,but are not limited to, thrombocytopenia and Scott syndrome.

Disorders involving T cells include, but are not limited to,cell-mediated hypersensitivity, such as delayed type hypersensitivityand T-cell-mediated cytotoxicity, and transplant rejection; autoimmunediseases, such as systemic lupus erythematosus, Sjögren syndrome,systemic sclerosis, inflammatory myopathies, mixed connective tissuedisease, and polyarteritis nodosa and other vasculitides; immunologicdeficiency syndromes, including but not limited to, primaryimmunodeficiencies, such as thymic hypoplasia, severe combinedimmunodeficiency diseases, and AIDS; leukopenia; reactive (inflammatory)proliferations of white cells, including but not limited to,leukocytosis, acute nonspecific lymphadenitis, and chronic nonspecificlymphadenitis; neoplastic proliferations of white cells, including butnot limited to lymphoid neoplasms, such as precursor T-cell neoplasms,such as acute lymphoblastic leukemia/lymphoma, peripheral T-cell andnatural killer cell neoplasms that include peripheral T-cell lymphoma,unspecified, adult T-cell leukemia/lymphoma, mycosis fungoides andSezary syndrome, and Hodgkin disease.

In normal bone marrow, the myelocytic series (polymorphonuclear cells)make up approximately 60% of the cellular elements, and the erythrocyticseries, 20-30%. Lymphocytes, monocytes, reticular cells, plasma cellsand megakaryocytes together constitute 10-20%. Lymphocytes make up 5-15%of normal adult marrow. In the bone marrow, cell types are add mixed sothat precursors of red blood cells (erythroblasts), macrophages(monoblasts), platelets (megakaryocytes), polymorphoneuclear leucocytes(myeloblasts), and lymphocytes (lymphoblasts) can be visible in onemicroscopic field. In addition, stem cells exist for the different celllineages, as well as a precursor stem cell for the committed progenitorcells of the different lineages. The various types of cells and stagesof each would be known to the person of ordinary skill in the art andare found, for example, on page 42 (FIG. 2-8) of Immunology,Imunopathology and Immunity, Fifth Edition, Sell et al. Simon andSchuster (1996), incorporated by reference for its teaching of celltypes found in the bone marrow. According, the invention is directed todisorders arising from these cells. These disorders include but are notlimited to the following: diseases involving hematopoietic stem cells;committed lymphoid progenitor cells; lymphoid cells including B andT-cells; committed myeloid progenitors, including monocytes,granulocytes, and megakaryocytes; and committed erythroid progenitors.These include but are not limited to the leukemias, including B-lymphoidleukemias, T-lymphoid leukemias, undifferentiated leukemias;erythroleukemia, megakaryoblastic leukemia, monocytic; [leukemias areencompassed with and without differentiation]; chronic and acutelymphoblastic leukemia, chronic and acute lymphocytic leukemia, chronicand acute myelogenous leukemia, lymphoma, myelo dysplastic syndrome,chronic and acute myeloid leukemia, myelomonocytic leukemia; chronic andacute myeloblastic leukemia, chronic and acute myelogenous leukemia,chronic and acute promyelocytic leukemia, chronic and acute myelocyticleukemia, hematologic malignancies of monocyte-macrophage lineage, suchas juvenile chronic myelogenous leukemia; secondary AML, antecedenthematological disorder; refractory anemia; aplastic anemia; reactivecutaneous angioendotheliomatosis; fibrosing disorders involving alteredexpression in dendritic cells, disorders including systemic sclerosis,E-M syndrome, epidemic toxic oil syndrome, eosinophilic fasciitislocalized forms of scleroderma, keloid, and fibrosing colonopathy;angiomatoid malignant fibrous histiocytoma; carcinoma, including primaryhead and neck squamous cell carcinoma; sarcoma, including kaposi'ssarcoma; fibroadenoma and phyllodes tumors, including mammaryfibroadenoma; stromal tumors; phyllodes tumors, including histiocytoma;erythroblastosis; neurofibromatosis; diseases of the vascularendothelium; demyelinating, particularly in old lesions; gliosis,vasogenic edema, vascular disease, Alzheimer's and Parkinson's disease;T-cell lymphomas; B-cell lymphomas.

Disorders involving the heart, include but are not limited to, heartfailure, including but not limited to, cardiac hypertrophy, left-sidedheart failure, and right-sided heart failure; ischemic heart disease,including but not limited to angina pectoris, myocardial infarction,chronic ischemic heart disease, and sudden cardiac death; hypertensiveheart disease, including but not limited to, systemic (left-sided)hypertensive heart disease and pulmonary (right-sided) hypertensiveheart disease; valvular heart disease, including but not limited to,valvular degeneration caused by calcification, such as calcific aorticstenosis, calcification of a congenitally bicuspid aortic valve, andmitral annular calcification, and myxomatous degeneration of the mitralvalve (mitral valve prolapse), rheumatic fever and rheumatic heartdisease, infective endocarditis, and noninfected vegetations, such asnonbacterial thrombotic endocarditis and endocarditis of systemic lupuserythematosus (Libman-Sacks disease), carcinoid heart disease, andcomplications of artificial valves; myocardial disease, including butnot limited to dilated cardiomyopathy, hypertrophic cardiomyopathy,restrictive cardiomyopathy, and myocarditis; pericardial disease,including but not limited to, pericardial effusion and hemopericardiumand pericarditis, including acute pericarditis and healed pericarditis,and rheumatoid heart disease; neoplastic heart disease, including butnot limited to, primary cardiac tumors, such as myxoma, lipoma,papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac effectsof noncardiac neoplasms; congenital heart disease, including but notlimited to, left-to-right shunts—late cyanosis, such as atrial septaldefect, ventricular septal defect, patent ductus arteriosus, andatrioventricular septal defect, right-to-left shunts—early cyanosis,such as tetralogy of fallot, transposition of great arteries, truncusarteriosus, tricuspid atresia, and total anomalous pulmonary venousconnection, obstructive congenital anomalies, such as coarctation ofaorta, pulmonary stenosis and atresia, and aortic stenosis and atresia,and disorders involving cardiac transplantation.

Disorders involving blood vessels include, but are not limited to,responses of vascular cell walls to injury, such as endothelialdysfunction and endothelial activation and intimal thickening; vasculardiseases including, but not limited to, congenital anomalies, such asarteriovenous fistula, atherosclerosis, and hypertensive vasculardisease, such as hypertension; inflammatory disease—the vasculitides,such as giant cell (temporal) arteritis, Takayasu arteritis,polyarteritis nodosa (classic), Kawasaki syndrome (mucocutaneous lymphnode syndrome), microscopic polyanglitis (microscopic polyarteritis,hypersensitivity or leukocytoclastic anglitis), Wegener granulomatosis,thromboanglitis obliterans (Buerger disease), vasculitis associated withother disorders, and infectious arteritis; Raynaud disease; aneurysmsand dissection, such as abdominal aortic aneurysms, syphilitic (luetic)aneurysms, and aortic dissection (dissecting hematoma); disorders ofveins and lymphatics, such as varicose veins, thrombophlebitis andphlebothrombosis, obstruction of superior vena cava (superior vena cavasyndrome), obstruction of inferior vena cava (inferior vena cavasyndrome), and lymphangitis and lymphedema; tumors, including benigntumors and tumor-like conditions, such as hemangioma, lymphangioma,glomus tumor (glomangioma), vascular ectasias, and bacillaryangiomatosis, and intermediate-grade (borderline low-grade malignant)tumors, such as Kaposi sarcoma and hemangloendothelioma, and malignanttumors, such as angiosarcoma and hemangiopericytoma; and pathology oftherapeutic interventions in vascular disease, such as balloonangioplasty and related techniques and vascular replacement, such ascoronary artery bypass graft surgery.

Disorders involving red cells include, but are not limited to, anemias,such as hemolytic anemias, including hereditary spherocytosis, hemolyticdisease due to erythrocyte enzyme defects: glucose-6-phosphatedehydrogenase deficiency, sickle cell disease, thalassemia syndromes,paroxysmal nocturnal hemoglobinuria, immunohemolytic anemia, andhemolytic anemia resulting from trauma to red cells; and anemias ofdiminished erythropoiesis, including megaloblastic anemias, such asanemias of vitamin B₁₂ deficiency: pernicious anemia, and anemia offolate deficiency, iron deficiency anemia, anemia of chronic disease,aplastic anemia, pure red cell aplasia, and other forms of marrowfailure.

Disorders involving B-cells include, but are not limited to precursorB-cell neoplasms, such as lymphoblastic leukemia/lymphoma. PeripheralB-cell neoplasms include, but are not limited to, chronic lymphocyticleukemia/small lymphocytic lymphoma, follicular lymphoma, diffuse largeB-cell lymphoma, Burkitt lymphoma, plasma cell neoplasms, multiplemyeloma, and related entities, lymphoplasmacytic lymphoma (Waldenstrommacroglobulinemia), mantle cell lymphoma, marginal zone lymphoma(MALToma), and hairy cell leukemia.

Disorders involving the liver include, but are not limited to, hepaticinjury; jaundice and cholestasis, such as bilirubin and bile formation;hepatic failure and cirrhosis, such as cirrhosis, portal hypertension,including ascites, portosystemic shunts, and splenomegaly; infectiousdisorders, such as viral hepatitis, including hepatitis A-E infectionand infection by other hepatitis viruses, clinicopathologic syndromes,such as the carrier state, asymptomatic infection, acute viralhepatitis, chronic viral hepatitis, and fulminant hepatitis; autoimmunehepatitis; drug- and toxin-induced liver disease, such as alcoholicliver disease; inborn errors of metabolism and pediatric liver disease,such as hemochromatosis, Wilson disease, α₁-antitrypsin deficiency, andneonatal hepatitis; intrahepatic biliary tract disease, such assecondary biliary cirrhosis, primary biliary cirrhosis, primarysclerosing cholangitis, and anomalies of the biliary tree; circulatorydisorders, such as impaired blood flow into the liver, including hepaticartery compromise and portal vein obstruction and thrombosis, impairedblood flow through the liver, including passive congestion andcentrilobular necrosis and peliosis hepatis, hepatic vein outflowobstruction, including hepatic vein thrombosis (Budd-Chiari syndrome)and veno-occlusive disease; hepatic disease associated with pregnancy,such as preeclampsia and eclampsia, acute fatty liver of pregnancy, andintrehepatic cholestasis of pregnancy; hepatic complications of organ orbone marrow transplantation, such as drug toxicity after bone marrowtransplantation, graft-versus-host disease and liver rejection, andnonimmunologic damage to liver allografts; tumors and tumorousconditions, such as nodular hyperplasias, adenomas, and malignanttumors, including primary carcinoma of the liver and metastatic tumors.

Disorders involving the brain include, but are not limited to, disordersinvolving neurons, and disorders involving glia, such as astrocytes,oligodendrocytes, ependymal cells, and microglia; cerebral edema, raisedintracranial pressure and herniation, and hydrocephalus; malformationsand developmental diseases, such as neural tube defects, forebrainanomalies, posterior fossa anomalies, and syringomyelia and hydromyelia;perinatal brain injury; cerebrovascular diseases, such as those relatedto hypoxia, ischemia, and infarction, including hypotension,hypoperfusion, and low-flow states—global cerebral ischemia and focalcerebral ischemia—infarction from obstruction of local blood supply,intracranial hemorrhage, including intracerebral (intraparenchymal)hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, andvascular malformations, hypertensive cerebrovascular disease, includinglacunar infarcts, slit hemorrhages, and hypertensive encephalopathy;infections, such as acute meningitis, including acute pyogenic(bacterial) meningitis and acute aseptic (viral) meningitis, acute focalsuppurative infections, including brain abscess, subdural empyema, andextradural abscess, chronic bacterial meningoencephalitis, includingtuberculosis and mycobacterioses, neurosyphilis, and neuroborreliosis(Lyme disease), viral meningoencephalitis, including arthropod-borne(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes simplexvirus Type 2, Varicalla-zoster virus (Herpes zoster), cytomegalovirus,poliomyelitis, rabies, and human immunodeficiency virus 1, includingHIV-1 meningoencephalitis (subacute encephalitis), vacuolar myelopathy,AIDS-associated myopathy, peripheral neuropathy, and AIDS in children,progressive multifocal leukoencephalopathy, subacute sclerosingpanencephalitis, fungal meningoencephalitis, other infectious diseasesof the nervous system; transmissible spongiform encephalopathies (priondiseases); demyelinating diseases, including multiple sclerosis,multiple sclerosis variants, acute disseminated encephalomyelitis andacute necrotizing hemorrhagic encephalomyelitis, and other diseases withdemyelination; degenerative diseases, such as degenerative diseasesaffecting the cerebral cortex, including Alzheimer disease and Pickdisease, degenerative diseases of basal ganglia and brain stem,including Parkinsonism, idiopathic Parkinson disease (paralysisagitans), progressive supranuclear palsy, corticobasal degeneration,multiple system atrophy, including striatonigral degenration, Shy-Dragersyndrome, and olivopontocerebellar atrophy, and Huntington disease;spinocerebellar degenerations, including spinocerebellar ataxias,including Friedreich ataxia, and ataxia-telanglectasia, degenerativediseases affecting motor neurons, including amyotrophic lateralsclerosis (motor neuron disease), bulbospinal atrophy (Kennedysyndrome), and spinal muscular atrophy; inborn errors of metabolism,such as leukodystrophies, including Krabbe disease, metachromaticleukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, andCanavan disease, mitochondrial encephalomyopathies, including Leighdisease and other mitochondrial encephalomyopathies; toxic and acquiredmetabolic diseases, including vitamin deficiencies such as thiamine(vitamin B₁) deficiency and vitamin B₁₂ deficiency, neurologic sequelaeof metabolic disturbances, including hypoglycemia, hyperglycemia, andhepatic encephatopathy, toxic disorders, including carbon monoxide,methanol, ethanol, and radiation, including combined methotrexate andradiation-induced injury; tumors, such as gliomas, includingastrocytoma, including fibrillary (diffuse) astrocytoma and glioblastomamultiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, andbrain stem glioma, oligodendroglioma, and ependymoma and relatedparaventricular mass lesions, neuronal tumors, poorly differentiatedneoplasms, including medulloblastoma, other parenchymal tumors,including primary brain lymphoma, germ cell tumors, and pinealparenchymal tumors, meningiomas, metastatic tumors, paraneoplasticsyndromes, peripheral nerve sheath tumors, including schwannoma,neurofibroma, and malignant peripheral nerve sheath tumor (malignantschwannoma), and neurocutaneous syndromes (phakomatoses), includingneurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindaudisease.

Disorders involving the ovary include, for example, polycystic ovariandisease, Stein-leventhal syndrome, Pseudomyxoma peritonei and stromalhyperthecosis; ovarian tumors such as, tumors of coelomic epithelium,serous tumors, mucinous tumors, endometeriod tumors, clear celladenocarcinoma, cystadenofibroma, brenner tumor, surface epithelialtumors; germ cell tumors such as mature (benign) teratomas, monodermalteratomas, immature malignant teratomas, dysgerminoma, endodermal sinustumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-thecacell tumors, thecoma-fibromas, androblastomas, hill cell tumors, andgonadoblastoma; and metastatic tumors such as Krukenberg tumors.

Disorders involving the kidney include, but are not limited to,congenital anomalies including, but not limited to, cystic diseases ofthe kidney, that include but are not limited to, cystic renal dysplasia,autosomal dominant (adult) polycystic kidney disease, autosomalrecessive (childhood) polycystic kidney disease, and cystic diseases ofrenal medulla, which include, but are not limited to, medullary spongekidney, and nephronophthisis-uremic medullary cystic disease complex,acquired (dialysis-associated) cystic disease, such as simple cysts;glomerular diseases including pathologies of glomerular injury thatinclude, but are not limited to, in situ immune complex deposition, thatincludes, but is not limited to, anti-GBM nephritis, Heymann nephritis,and antibodies against planted antigens, circulating immune complexnephritis, antibodies to glomerular cells, cell-mediated immunity inglomerulonephritis, activation of alternative complement pathway,epithelial cell injury, and pathologies involving mediators ofglomerular injury including cellular and soluble mediators, acuteglomerulonephritis, such as acute proliferative (poststreptococcal,postinfectious) glomerulonephritis, including but not limited to,poststreptococcal glomerulonephritis and nonstreptococcal acuteglomerulonephritis, rapidly progressive (crescentic) glomerulonephritis,nephrotic syndrome, membranous glomerulonephritis (membranousnephropathy), minimal change disease (lipoid nephrosis), focal segmentalglomerulosclerosis, membranoproliferative glomerulonephritis, IgAnephropathy (Berger disease), focal proliferative and necrotizingglomerulonephritis (focal glomerulonephritis), hereditary nephritis,including but not limited to, Alport syndrome and thin membrane disease(benign familial hematuria), chronic glomerulonephritis, glomerularlesions associated with systemic disease, including but not limited to,systemic lupus erythematosus, Henoch-Schönlein purpura, bacterialendocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary andimmunotactoid glomerulonephritis, and other systemic disorders; diseasesaffecting tubules and interstitium, including acute tubular necrosis andtubulointerstitial nephritis, including but not limited to,pyelonephritis and urinary tract infection, acute pyelonephritis,chronic pyelonephritis and reflux nephropathy, and tubulointerstitialnephritis induced by drugs and toxins, including but not limited to,acute drug-induced interstitial nephritis, analgesic abuse nephropathy,nephropathy associated with nonsteroidal anti-inflammatory drugs, andother tubulointerstitial diseases including, but not limited to, uratenephropathy, hypercalcemia and nephrocalcinosis, and multiple myeloma;diseases of blood vessels including benign nephrosclerosis, malignanthypertension and accelerated nephrosclerosis, renal artery stenosis, andthrombotic microangiopathies including, but not limited to, classic(childhood) hemolytic-uremic syndrome, adult hemolytic-uremicsyndrome/thrombotic thrombocytopenic purpura, idiopathic HUS/TTP, andother vascular disorders including, but not limited to, atheroscleroticischemic renal disease, atheroembolic renal disease, sickle cell diseasenephropathy, diffuse cortical necrosis, and renal infarcts; urinarytract obstruction (obstructive uropathy); urolithiasis (renal calculi,stones); and tumors of the kidney including, but not limited to, benigntumors, such as renal papillary adenoma, renal fibroma or hamartoma(renomedullary interstitial cell tumor), angiomyolipoma, and oncocytoma,and malignant tumors, including renal cell carcinoma (hypemephroma,adenocarcinoma of kidney), which includes urothelial carcinomas of renalpelvis.

Disorders involving the skeletal muscle include tumors such asrhabdomyosarcoma.

Bone-forming cells include the osteoprogenitor cells, osteoblasts, andosteocytes. The disorders of the bone are complex because they may havean impact on the skeleton during any of its stages of development.Hence, the disorders may have variable manifestations and may involveone, multiple or all bones of the body. Such disorders include,congenital malformations, achondroplasia and thanatophoric dwarfism,diseases associated with abnormal matrix such as type 1 collagendisease, osteoporosis, Paget disease, rickets, osteomalacia,high-turnover osteodystrophy, low-turnover of aplastic disease,osteonecrosis, pyogenic osteomyelitis, tuberculous osteomyelitism,osteoma, osteoid osteoma, osteoblastoma, osteosarcoma, osteochondroma,chondromas, chondroblastoma, chondromyxoid fibroma, chondrosarcoma,fibrous cortical defects, fibrous dysplasia, fibrosarcoma, malignantfibrous histiocytoma, Ewing sarcoma, primitive neuroectodermal tumor,giant cell tumor, and metastatic tumors.

Disorders involving the pancreas include those of the exocrine pancreassuch as congenital anomalies, including but not limited to, ectopicpancreas; pancreatitis, including but not limited to, acutepancreatitis; cysts, including but not limited to, pseudocysts; tumors,including but not limited to, cystic tumors and carcinoma of thepancreas; and disorders of the endocrine pancreas such as, diabetesmellitus; islet cell tumors, including but not limited to, insulinomas,gastrinomas, and other rare islet cell tumors.

Diseases of the skin, include but are not limited to, disorders ofpigmentation and melanocytes, including but not limited to, vitiligo,freckle, melasma, lentigo, nevocellular nevus, dysplastic nevi, andmalignant melanoma; benign epithelial tumors, including but not limitedto, seborrheic keratoses, acanthosis nigricans, fibroepithelial polyp,epithelial cyst, keratoacanthoma, and adnexal (appendage) tumors;premalignant and malignant epidermal tumors, including but not limitedto, actinic keratosis, squamous cell carcinoma, basal cell carcinoma,and merkel cell carcinoma; tumors of the dermis, including but notlimited to, benign fibrous histiocytoma, dermatofibrosarcomaprotuberans, xanthomas, and dermal vascular tumors; tumors of cellularimmigrants to the skin, including but not limited to, histiocytosis X,mycosis fungoides (cutaneous T-cell lymphoma), and mastocytosis;disorders of epidermal maturation, including but not limited to,ichthyosis; acute inflammatory dermatoses, including but not limited to,urticaria, acute eczematous dermatitis, and erythema multiforme; chronicinflammatory dermatoses, including but not limited to, psoriasis, lichenplanus, and lupus erythematosus; blistering (bullous) diseases,including but not limited to, pemphigus, bullous pemphigoid, dermatitisherpetiformis, and noninflammatory blistering diseases: epidermolysisbullosa and porphyria; disorders of epidermal appendages, including butnot limited to, acne vulgaris; panniculitis, including but not limitedto, erythema nodosum and erythema induratum; and infection andinfestation, such as verrucae, molluscum contagiosum, impetigo,superficial fungal infections, and arthropod bites, stings, andinfestations.

Disorders of the breast include, but are not limited to, disorders ofdevelopment; inflammations, including but not limited to, acutemastitis, periductal mastitis, periductal mastitis (recurrent subareolarabscess, squamous metaplasia of lactiferous ducts), mammary ductectasia, fat necrosis, granulomatous mastitis, and pathologiesassociated with silicone breast implants; fibrocystic changes;proliferative breast disease including, but not limited to, epithelialhyperplasia, sclerosing adenosis, and small duct papillomas; tumorsincluding, but not limited to, stromal tumors such as fibroadenoma,phyllodes tumor, and sarcomas, and epithelial tumors such as large ductpapilloma; carcinoma of the breast including in situ (noninvasive)carcinoma that includes ductal carcinoma in situ (including Paget'sdisease) and lobular carcinoma in situ, and invasive (infiltrating)carcinoma including, but not limited to, invasive ductal carcinoma, nospecial type, invasive lobular carcinoma, medullary carcinoma, colloid(mucinous) carcinoma, tubular carcinoma, and invasive papillarycarcinoma, and miscellaneous malignant neoplasms.

Disorders in the male breast include, but are not limited to,gynecomastia and carcinoma.

Disorders involving the prostate include, but are not limited to,inflammations, benign enlargement, for example, nodular hyperplasia(benign prostatic hypertrophy or hyperplasia), and tumors such ascarcinoma.

Disorders involving the colon include, but are not limited to,congenital anomalies, such as atresia and stenosis, Meckel diverticulum,congenital aganglionic megacolon-Hirschsprung disease; enterocolitis,such as diarrhea and dysentery, infectious enterocolitis, includingviral gastroenteritis, bacterial enterocolitis, necrotizingenterocolitis, antibiotic-associated colitis (pseudomembranous colitis),and collagenous and lymphocytic colitis, miscellaneous intestinalinflammatory disorders, including parasites and protozoa, acquiredimmunodeficiency syndrome, transplantation, drug-induced intestinalinjury, radiation enterocolitis, neutropenic colitis (typhlitis), anddiversion colitis; idiopathic inflammatory bowel disease, such as Crohndisease and ulcerative colitis; tumors of the colon, such asnon-neoplastic polyps, adenomas, familial syndromes, colorectalcarcinogenesis, colorectal carcinoma, and carcinoid tumors.

Disorders involving the lung include, but are not limited to, congenitalanomalies; atelectasis; diseases of vascular origin, such as pulmonarycongestion and edema, including hemodynamic pulmonary edema and edemacaused by microvascular injury, adult respiratory distress syndrome(diffuse alveolar damage), pulmonary embolism, hemorrhage, andinfarction, and pulmonary hypertension and vascular sclerosis; chronicobstructive pulmonary disease, such as emphysema, chronic bronchitis,bronchial asthma, and bronchiectasis; diffuse interstitial(infiltrative, restrictive) diseases, such as pneumoconioses,sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitialpneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia(pulmonary infiltration with eosinophilia), Bronchiolitisobliterans-organizing pneumonia, diffuse pulmonary hemorrhage syndromes,including Goodpasture syndrome, idiopathic pulmonary hemosiderosis andother hemorrhagic syndromes, pulmonary involvement in collagen vasculardisorders, and pulmonary alveolar proteinosis; complications oftherapies, such as drug-induced lung disease, radiation-induced lungdisease, and lung transplantation; tumors, such as bronchogeniccarcinoma, including paraneoplastic syndromes, bronchioloalveolarcarcinoma, neuroendocrine tumors, such as bronchial carcinoid,miscellaneous tumors, and metastatic tumors; pathologies of the pleura,including inflammatory pleural effusions, noninflammatory pleuraleffusions, pneumothorax, and pleural tumors, including solitary fibroustumors (pleural fibroma) and malignant mesothelioma.

Disorders involving the spleen include, but are not limited to,splenomegaly, including nonspecific acute splenitis, congestivespenomegaly, and spenic infarcts; neoplasms, congenital anomalies, andrupture. Disorders associated with splenomegaly include infections, suchas nonspecific splenitis, infectious mononucleosis, tuberculosis,typhoid fever, brucellosis, cytomegalovirus, syphilis, malaria,histoplasmosis, toxoplasmosis, kala-azar, trypanosomiasis,schistosomiasis, leishmaniasis, and echinococcosis; congestive statesrelated to partial hypertension, such as cirrhosis of the liver, portalor splenic vein thrombosis, and cardiac failure; lymphohematogenousdisorders, such as Hodgkin disease, non-Hodgkin lymphomas/leukemia,multiple myeloma, myeloproliferative disorders, hemolytic anemias, andthrombocytopenic purpura; immunologic-inflammatory conditions, such asrheumatoid arthritis and systemic lupus erythematosus; storage diseasessuch as Gaucher disease, Niemann-Pick disease, andmucopolysaccharidoses; and other conditions, such as amyloidosis,primary neoplasms and cysts, and secondary neoplasms.

Disorders involving the thymus include developmental disorders, such asDiGeorge syndrome with thymic hypoplasia or aplasia; thymic cysts;thymic hypoplasia, which involves the appearance of lymphoid follicleswithin the thymus, creating thymic follicular hyperplasia; and thymomas,including germ cell tumors, lynphomas, Hodgkin disease, and carcinoids.Thymomas can include benign or encapsulated thymoma, and malignantthymoma Type I (invasive thymoma) or Type II, designated thymiccarcinoma.

Disorders involving the tonsils include, but are not limited to,tonsillitis, Peritonsillar abscess, squamous cell carcinoma, dyspnea,hyperplasia, follicular hyperplasia, reactive lymphoid hyperplasia,non-Hodgkin's lymphoma and B-cell lymphoma.

A novel human phospholipid scramblase-like gene sequence, referred to as32621, is provided. This gene sequence and variants and fragmentsthereof are encompassed by the term “phospholipid scramblase-like”molecules or sequences as used herein. The phospholipid scramblase-likesequences find use in modulating a phospholipid scramblase function. By“modulating” is intended the upregulating or downregulating of aresponse. That is, the compositions of the invention affect the targetactivity in either a positive or negative fashion. The sequences of theinvention find use in modulating the immune, hematopoiesis, bloodcoagulation, and plasma clotting systems.

The human phospholipid scramblase-like gene, clone 32621 was identifiedin a human primary osteoblast cDNA library. Clone 32621 encodes an mRNAtranscript having the corresponding cDNA set forth in SEQ ID NO:16. Thistranscript has a 990 nucleotide open reading frame (nucleotides 156-1142of SEQ ID NO:16; SEQ ID NO:18), which encodes a 329 amino acid protein(SEQ ID NO:17). A transmembrane segment from amino acids (aa) 304-320was predicted by MEMSAT. Prosite program analysis was used to predictvarious sites within the h32621 protein. N-glycosylation sites werepredicted at aa 18-21, 92-95, and 147-150. Protein kinase Cphosphorylation sites were predicted at aa 170-172 and 204-206. Caseinkinase II phosphorylation sites were predicted at aa 7-10, 135-138, and259-262. A tyrosine kinase phosphorylation site was predicted at aa146-154. N-myristoylation sites were predicted at aa 3-8, 55-60,216-221, and 281-286.

The 32621 protein shares approximately 45% identity to the Mus musculusphospholipid scramblase-like and approximately 41% identity to the Homosapiens hMmTRA1b protein as determined by pairwise alignment (FIG. 21).

The 32621 protein displays approximately 47% identity from aa 206-321 toa ProDom consensus sequence found in murine phospholipid scramblase-like1; approximately 38% identity from aa 131-190 to a ProDom consensussequence found in human phospholipid scramblase-like (MmTRA1b);approximately 59% identity from aa 108-129 to a ProDom consensussequence found in murine phospholipid scramblase-like 1, human MmTRA1b,and murine transplantability associated protein I (TRA1); and,approximately 38% identity from aa 59-111 to a ProDom consensus sequencefound in murine SRG3 and human BAF155. Phospholipid scramblase-like 1 isa plasma membrane protein that mediates accelerated transbilayermigration of phospholipids upon binding calcium ions. See for example,Zhou et al. (1998) Biochemistry 37:2356-2360. The plasma membraneprotein, human phospholipid scramblase-like, also mediates transbilayermigration of phospholipids upon Ca²⁺ binding. The human scramblase mayplay a central role in the initiation of fibrin clot formation and inthe recognition of apoptotic and injured cells by thereticuloendothelial system. Defects or deficiency of this scramblasecauses Scott syndrome and possibly other bleeding disorders. See, forexample, Zho et al. (1997) J. Biol. Chem. 272:18240-18244, Kasukabe etal. (1998) Biochem. Biophys. Res. Commun. 249:449-455, Basse et al.(1996) J. Biol. Chem. 271:17205-17210, and Zhou et al. (1998)Biochemistry 37:2356-2360. Murine SRG3 belongs to a family of SWI/SNFrelated, matrix associated, actin dependent regulator of chromatinassembly. Human BAF155 is the 155 kDa subunit of the SWI/SNF complex(Wang et al. (1996) Genes and Dev. 10:2117-2130). The sequences wereidentified by the ProDom program, which is available from INRA, GREG(107/94), MESR (ACC-SV13), the CNRS “Genome Initiative” and the EuropeanUnion. The ProDom Program (http://www.toulouse.inra.fr/prodom.html)allows analysis of domain arrangements in proteins and protein families.A detailed description of ProDom analysis can be found in Corpet et al.(1999) Nuc. Acids Res. 27:263-267.

The human phospholipid scramblase-like sequences of the invention aremembers of a family of molecules (PL flip/flop genes). The term “family”when referring to the proteins and nucleic acid molecules of theinvention is intended to mean two or more proteins or nucleic acidmolecules having sufficient amino acid or nucleotide sequence identityas defined herein. Such family members can be naturally occurring andcan be from either the same or different species. For example, a familycan contain a first protein of murine origin and a homologue of thatprotein of human origin, as well as a second, distinct protein of humanorigin and a murine homologue of that protein. Members of a family mayalso have common functional characteristics.

Preferred human phospholipid scramblase-like polypeptides of the presentinvention have an amino acid sequence sufficiently identical to theamino acid sequence of SEQ ID NO:17. The term “sufficiently identical”is used herein to refer to a first amino acid or nucleotide sequencethat contains a sufficient or minimum number of identical or equivalent(e.g., with a similar side chain) amino acid residues or nucleotides toa second amino acid or nucleotide sequence such that the first andsecond amino acid or nucleotide sequences have a common structuraldomain and/or common functional activity. For example, amino acid ornucleotide sequences that contain a common structural domain having atleast about 60%, 65%, 70%, 75% 85%, 90%, 95%, 96%, 97%, 98% or 99%identity are defined herein as sufficiently identical.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. The percent identity between two sequences can bedetermined using techniques similar to those described below, with orwithout allowing gaps. In calculating percent identity, typically exactmatches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. In a preferred embodiment,the percent identity between two amino acid sequences is determinedusing the Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453 algorithmwhich has been incorporated into the GAP program in the GCG softwarepackage (available at http://www.gcg.com), using either a Blossum 62matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferredembodiment, the percent identity between two nucleotide sequences isdetermined using the GAP program in the GCG software package (availableat http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused if the practitioner is uncertain about what parameters should beapplied to determine if a molecule is within a sequence identity orhomology limitation of the invention) is using a Blossum 62 scoringmatrix with a gap open penalty of 12, a gap extend penalty of 4, and aframeshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of Karlin and Altschul (1990) Proc.Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al.(1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performedwith the NBLAST program, score=100, wordlength=12, to obtain nucleotidesequences homologous to 32621-like nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3, to obtain amino acid sequenceshomologous to 32621-like protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-Blast can be used to perform an iterated search thatdetects distant relationships between molecules. See Altschul et al.(1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blastprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Anotherpreferred, non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Myers and Miller (1988)CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program(version 2.0), which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

Accordingly, another embodiment of the invention features isolated humanphospholipid scramblase-like proteins and polypeptides having a humanphospholipid scramblase-like protein activity. As used interchangeablyherein, a “human phospholipid scramblase-like protein activity”,“biological activity of a human phospholipid scramblase-like protein”,or “functional activity of a human phospholipid scramblase-like protein”refers to an activity exerted by a human phospholipid scramblase-likeprotein, polypeptide, or nucleic acid molecule on a human phospholipidscramblase-like responsive cell as determined in vivo, or in vitro,according to standard assay techniques. A human phospholipidscramblase-like activity can be a direct activity, such as anassociation with or an enzymatic activity on a second protein, or anindirect activity, such as a cellular signaling activity mediated byinteraction of the human phospholipid scramblase-like protein with asecond protein. In a preferred embodiment, a human phospholipidscramblase-like activity includes at least one or more of the followingactivities: modulating (stimulating and/or enhancing or inhibiting)phospholipid redistribution in the plasma membrane.

An “isolated” or “purified” human phospholipid scramblase-like nucleicacid molecule or protein, or biologically active portion thereof, issubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. Preferably,an “isolated” nucleic acid is free of sequences (preferably proteinencoding sequences) that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forpurposes of the invention, “isolated” when used to refer to nucleic acidmolecules excludes isolated chromosomes. For example, in variousembodiments, the isolated human phospholipid scramblase-like nucleicacid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank thenucleic acid molecule in genomic DNA of the cell from which the nucleicacid is derived. A human phospholipid scramblase-like protein that issubstantially free of cellular material includes preparations of humanphospholipid scramblase-like protein having less than about 30%, 20%,10%, or 5% (by dry weight) of non-human phospholipid scramblase-likeprotein (also referred to herein as a “contaminating protein”). When thehuman phospholipid scramblase-like protein or biologically activeportion thereof is recombinantly produced, preferably, culture mediumrepresents less than about 30%, 20%, 10%, or 5% of the volume of theprotein preparation. When human phospholipid scramblase-like protein isproduced by chemical synthesis, preferably the protein preparations haveless than about 30%, 20%, 10%, or 5% (by dry weight) of chemicalprecursors or non-human phospholipid scramblase-like chemicals.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculescomprising nucleotide sequences encoding human phospholipidscramblase-like proteins and polypeptides or biologically activeportions thereof, as well as nucleic acid molecules sufficient for useas hybridization probes to identify human phospholipidscramblase-like-encoding nucleic acids (e.g., human phospholipidscramblase-like mRNA) and fragments for use as PCR primers for theamplification or mutation of human phospholipid scramblase-like nucleicacid molecules. As used herein, the term “nucleic acid molecule” isintended to include DNA molecules (e.g., cDNA or genomic DNA) and RNAmolecules (e.g., mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

Nucleotide sequences encoding the human phospholipid scramblase-likeproteins of the present invention include sequences set forth in SEQ IDNO:17 and complements thereof. By “complement” is intended a nucleotidesequence that is sufficiently complementary to a given nucleotidesequence such that it can hybridize to the given nucleotide sequence tothereby form a stable duplex. The corresponding amino acid sequence forthe human phospholipid scramblase-like protein encoded by thesenucleotide sequences is set forth in SEQ ID NO:16. The invention alsoencompasses nucleic acid molecules comprising nucleotide sequencesencoding partial-length human phospholipid scramblase-like proteins,including the sequence set forth in SEQ ID NO:17, and complementsthereof.

Nucleic acid molecules that are fragments of these human phospholipidscramblase-like nucleotide sequences are also encompassed by the presentinvention. By “fragment” is intended a portion of the nucleotidesequence encoding a human phospholipid scramblase-like protein. Afragment of a human phospholipid scramblase-like nucleotide sequence mayencode a biologically active portion of a human phospholipidscramblase-like protein, or it may be a fragment that can be used as ahybridization probe or PCR primer using methods disclosed below. Abiologically active portion of a human phospholipid scramblase-likeprotein can be prepared by isolating a portion of one of the humanphospholipid scramblase-like nucleotide sequences of the invention,expressing the encoded portion of the human phospholipid scramblase-likeprotein (e.g., by recombinant expression in vitro), and assessing theactivity of the encoded portion of the human phospholipidscramblase-like protein. Nucleic acid molecules that are fragments of ahuman phospholipid scramblase-like nucleotide sequence comprise at least15, 20, 50, 75, 100, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1500 nucleotides, or up to the number of nucleotides present in afull-length human phospholipid scramblase-like nucleotide sequencedisclosed herein (for example, 1542 nucleotides for SEQ ID NO:16)depending upon the intended use. Alternatively, a nucleic acid moleculesthat is a fragment of an 32621-like nucleotide sequence of the presentinvention comprises a nucleotide sequence consisting of nucleotides1-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800,800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400,1400-1500, 1500-1542 of SEQ ID NO:16 or 18.

It is understood that isolated fragments include any contiguous sequencenot disclosed prior to the invention as well as sequences that aresubstantially the same and which are not disclosed. Accordingly, if anisolated fragment is disclosed prior to the present invention, thatfragment is not intended to be encompassed by the invention. When asequence is not disclosed prior to the present invention, an isolatednucleic acid fragment is at least about 12, 15, 20, 25, or 30 contiguousnucleotides. Other regions of the nucleotide sequence may comprisefragments of various sizes, depending upon potential homology withpreviously disclosed sequences.

A fragment of a human phospholipid scramblase-like nucleotide sequencethat encodes a biologically active portion of a human phospholipidscramblase-like protein of the invention will encode at least 15, 25,30, 50, 75, 100, 125, 150, 175, 200, 250, or 300 contiguous amino acids,or up to the total number of amino acids present in a full-length humanphospholipid scramblase-like protein of the invention (for example, 329amino acids for SEQ ID NO:17). Alternatively, a fragment of apolypeptide of the present invention comprises an amino acid sequenceconsisting of amino acid residues 1-20, 20-40, 40-60, 60-80, 80-100,100-120, 120-140, 140-160, 160-180, 180-200, 200-220, 220-240, 240-260,260-280, 280-300, 300-320, 320-329 of SEQ ID NO:17. Fragments of a humanphospholipid scramblase-like nucleotide sequence that are useful ashybridization probes for PCR primers generally need not encode abiologically active portion of a human phospholipid scramblase-likeprotein.

Nucleic acid molecules that are variants of the human phospholipidscramblase-like nucleotide sequences disclosed herein are alsoencompassed by the present invention. “Variants” of the humanphospholipid scramblase-like nucleotide sequences include thosesequences that encode the human phospholipid scramblase-like proteinsdisclosed herein but that differ conservatively because of thedegeneracy of the genetic code. These naturally occurring allelicvariants can be identified with the use of well-known molecular biologytechniques, such as polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant nucleotide sequences also includesynthetically derived nucleotide sequences that have been generated, forexample, by using site-directed mutagenesis but which still encode thehuman phospholipid scramblase-like proteins disclosed in the presentinvention as discussed below. Generally, nucleotide sequence variants ofthe invention will have at least 45%, 55%, 65%, 75%, 85%, 95%, or 98%identity to a particular nucleotide sequence disclosed herein. A varianthuman phospholipid scramblase-like nucleotide sequence will encode ahuman phospholipid scramblase-like protein that has an amino acidsequence having at least 45%, 55%, 65%, 75%, 85%, 95%, or 98% identityto the amino acid sequence of a human phospholipid scramblase-likeprotein disclosed herein.

In addition to the human phospholipid scramblase-like nucleotidesequences shown in SEQ ID NO:16 it will be appreciated by those skilledin the art that DNA sequence polymorphisms that lead to changes in theamino acid sequences of human phospholipid scramblase-like proteins mayexist within a population (e.g., the human population). Such geneticpolymorphism in a human phospholipid scramblase-like gene may existamong individuals within a population due to natural allelic variation.An allele is one of a group of genes that occur alternatively at a givengenetic locus. As used herein, the terms “gene” and “recombinant gene”refer to nucleic acid molecules comprising an open reading frameencoding a human phospholipid scramblase-like protein, preferably amammalian human phospholipid scramblase-like protein. As used herein,the phrase “allelic variant” refers to a nucleotide sequence that occursat a human phospholipid scramblase-like locus or to a polypeptideencoded by the nucleotide sequence. Such natural allelic variations cantypically result in 1-5% variance in the nucleotide sequence of thehuman phospholipid scramblase-like gene. Any and all such nucleotidevariations and resulting amino acid polymorphisms or variations in ahuman phospholipid scramblase-like sequence that are the result ofnatural allelic variation and that do not alter the functional activityof human phospholipid scramblase-like proteins are intended to be withinthe scope of the invention.

Moreover, nucleic acid molecules encoding human phospholipidscramblase-like proteins from other species (human phospholipidscramblase-like homologues), which have a nucleotide sequence differingfrom that of the human phospholipid scramblase-like sequences disclosedherein, are intended to be within the scope of the invention. Forexample, nucleic acid molecules corresponding to natural allelicvariants and homologues of the human phospholipid scramblase-like cDNAof the invention can be isolated based on their identity to the humanphospholipid scramblase-like nucleic acid disclosed herein using thehuman cDNA, or a portion thereof, as a hybridization probe according tostandard hybridization techniques under stringent hybridizationconditions as disclosed below.

In addition to naturally-occurring allelic variants of the humanphospholipid scramblase-like sequences that may exist in the population,the skilled artisan will further appreciate that changes can beintroduced by mutation into the nucleotide sequences of the inventionthereby leading to changes in the amino acid sequence of the encodedhuman phospholipid scramblase-like proteins, without altering thebiological activity of the human phospholipid scramblase-like proteins.Thus, an isolated nucleic acid molecule encoding a human phospholipidscramblase-like protein having a sequence that differs from that of SEQID NO:16 can be created by introducing one or more nucleotidesubstitutions, additions, or deletions into the corresponding nucleotidesequence disclosed herein, such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein. Mutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Such variantnucleotide sequences are also encompassed by the present invention.

For example, preferably, conservative amino acid substitutions may bemade at one or more predicted, preferably nonessential amino acidresidues. A “nonessential” amino acid residue is a residue that can bealtered from the wild-type sequence of a human phospholipidscramblase-like protein (e.g., the sequence of SEQ ID NO:17) withoutaltering the biological activity, whereas an “essential” amino acidresidue is required for biological activity. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Such substitutions would not bemade for conserved amino acid residues, or for amino acid residuesresiding within a conserved motif, such as the growth factor andcytokine receptor signature 2 sequence and the U-PAR/Ly-6 domainsequence of SEQ ID NO:17, where such residues are essential for proteinactivity.

Alternatively, variant human phospholipid scramblase-like nucleotidesequences can be made by introducing mutations randomly along all orpart of a human phospholipid scramblase-like coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forhuman phospholipid scramblase-like biological activity to identifymutants that retain activity. Following mutagenesis, the encoded proteincan be expressed recombinantly, and the activity of the protein can bedetermined using standard assay techniques.

Thus the nucleotide sequences of the invention include the sequencesdisclosed herein as well as fragments and variants thereof. The humanphospholipid scramblase-like nucleotide sequences of the invention, andfragments and variants thereof, can be used as probes and/or primers toidentify and/or clone human phospholipid scramblase-like homologues inother cell types, e.g., from other tissues, as well as humanphospholipid scramblase-like homologues from other mammals. Such probescan be used to detect transcripts or genomic sequences encoding the sameor identical proteins. These probes can be used as part of a diagnostictest kit for identifying cells or tissues that misexpress a humanphospholipid scramblase-like protein, such as by measuring levels of ahuman phospholipid scramblase-like-encoding nucleic acid in a sample ofcells from a subject, e.g., detecting human phospholipid scramblase-likemRNA levels or determining whether a genomic human phospholipidscramblase-like gene has been mutated or deleted.

In this manner, methods such as PCR, hybridization, and the like can beused to identify such sequences having substantial identity to thesequences of the invention. See, for example, Sambrook et al. (1989)Molecular Cloning: Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.) and Innis, et al. (1990) PCRProtocols: A Guide to Methods and Applications (Academic Press, NY).Human phospholipid scramblase-like nucleotide sequences isolated basedon their sequence identity to the human phospholipid scramblase-likenucleotide sequences set forth herein or to fragments and variantsthereof are encompassed by the present invention.

In a hybridization method, all or part of a known human phospholipidscramblase-like nucleotide sequence can be used to screen cDNA orgenomic libraries. Methods for construction of such cDNA and genomiclibraries are generally known in the art and are disclosed in Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., ColdSpring Harbor Laboratory Press, Plainview, N.Y.). The so-calledhybridization probes may be genomic DNA fragments, cDNA fragments, RNAfragments, or other oligonucleotides, and may be labeled with adetectable group such as ³²P, or any other detectable marker, such asother radioisotopes, a fluorescent compound, an enzyme, or an enzymeco-factor. Probes for hybridization can be made by labeling syntheticoligonucleotides based on the known human phospholipid scramblase-likenucleotide sequence disclosed herein. Degenerate primers designed on thebasis of conserved nucleotides or amino acid residues in a known humanphospholipid scramblase-like nucleotide sequence or encoded amino acidsequence can additionally be used. The probe typically comprises aregion of nucleotide sequence that hybridizes under stringent conditionsto at least about 12, preferably about 25, more preferably about 50, 75,100, 125, 150, 175, 200, 250, 300, 350, or 400 consecutive nucleotidesof a human phospholipid scramblase-like nucleotide sequence of theinvention or a fragment or variant thereof. Preparation of probes forhybridization is generally known in the art and is disclosed in Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., ColdSpring Harbor Laboratory Press, Plainview, N.Y.), herein incorporated byreference.

For example, in one embodiment, a previously unidentified humanphospholipid scramblase-like nucleic acid molecule hybridizes understringent conditions to a probe that is a nucleic acid moleculecomprising one of the human phospholipid scramblase-like nucleotidesequences of the invention or a fragment thereof. In another embodiment,the previously unknown human phospholipid scramblase-like nucleic acidmolecule is at least 300, 325, 350, 375, 400, 425, 450, 500, 550, 600,650, 700, 800, 900, 1000, 2,000, 3,000, 4,000 or 5,000 nucleotides inlength and hybridizes under stringent conditions to a probe that is anucleic acid molecule comprising one of the human phospholipidscramblase-like nucleotide sequences disclosed herein or a fragmentthereof.

Accordingly, in another embodiment, an isolated previously unknown humanphospholipid scramblase-like nucleic acid molecule of the invention isat least 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700,800, 900, 1000, 1,100, 1,200, 1,300, or 1,400 nucleotides in length andhybridizes under stringent conditions to a probe that is a nucleic acidmolecule comprising one of the nucleotide sequences of the invention,preferably the coding sequence set forth in SEQ ID NO:17 or acomplement, fragment, or variant thereof.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences typically remain hybridized to each other.Such stringent conditions are known to those skilled in the art and canbe found in Current Protocols in Molecular Biology (John Wiley & Sons,New York (1989)), 6.3.1-6.3.6. A preferred, example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50° C. Another example of stringent hybridizationconditions are hybridization in 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at55° C. A further example of stringent hybridization conditions arehybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.Preferably, stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 65° C. Particularly preferredstringency conditions (and the conditions that should be used if thepractitioner is uncertain about what conditions should be applied todetermine if a molecule is within a hybridization limitation of theinvention) are 0.5M Sodium Phosphate, 7% SDS at 65° C., followed by oneor more washes at 0.2×SSC, 1% SDS at 65° C. Preferably, an isolatednucleic acid molecule that hybridizes under stringent conditions to a32621-like sequence of the invention corresponds to anaturally-occurring nucleic acid molecule. As used herein, a“naturally-occurring” nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural protein).

Thus, in addition to the human phospholipid scramblase-like nucleotidesequences disclosed herein and fragments and variants thereof, theisolated nucleic acid molecules of the invention also encompasshomologous DNA sequences identified and isolated from other cells and/ororganisms by hybridization with entire or partial sequences obtainedfrom the human phospholipid scramblase-like nucleotide sequencesdisclosed herein or variants and fragments thereof.

The present invention also encompasses antisense nucleic acid molecules,i.e., molecules that are complementary to a sense nucleic acid encodinga protein, e.g., complementary to the coding strand of a double-strandedcDNA molecule, or complementary to an mRNA sequence. Accordingly, anantisense nucleic acid can hydrogen bond to a sense nucleic acid. Theantisense nucleic acid can be complementary to an entire humanphospholipid scramblase-like coding strand, or to only a portionthereof, e.g., all or part of the protein coding region (or open readingframe). An antisense nucleic acid molecule can be antisense to anoncoding region of the coding strand of a nucleotide sequence encodinga human phospholipid scramblase-like protein. The noncoding regions arethe 5′ and 3′ sequences that flank the coding region and are nottranslated into amino acids.

Given the coding-strand sequence encoding a human phospholipidscramblase-like protein disclosed herein (e.g., SEQ ID NO:17), antisensenucleic acids of the invention can be designed according to the rules ofWatson and Crick base pairing. The antisense nucleic acid molecule canbe complementary to the entire coding region of human phospholipidscramblase-like mRNA, but more preferably is an oligonucleotide that isantisense to only a portion of the coding or noncoding region of humanphospholipid scramblase-like mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of human phospholipid scramblase-like mRNA. Anantisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45, or 50 nucleotides in length. An antisense nucleic acidof the invention can be constructed using chemical synthesis andenzymatic ligation procedures known in the art.

For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, including, but not limited to, for example e.g., phosphorothioatederivatives and acridine substituted nucleotides. Alternatively, theantisense nucleic acid can be produced biologically using an expressionvector into which a nucleic acid has been subcloned in an antisenseorientation (i.e., RNA transcribed from the inserted nucleic acid willbe of an antisense orientation to a target nucleic acid of interest,described further in the following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a humanphospholipid scramblase-like protein to thereby inhibit expression ofthe protein, e.g., by inhibiting transcription and/or translation. Anexample of a route of administration of antisense nucleic acid moleculesof the invention includes direct injection at a tissue site.Alternatively, antisense nucleic acid molecules can be modified totarget selected cells and then administered systemically. For example,antisense molecules can be linked to peptides or antibodies to form acomplex that specifically binds to receptors or antigens expressed on aselected cell surface. The antisense nucleic acid molecules can also bedelivered to cells using the vectors described herein. To achievesufficient intracellular concentrations of the antisense molecules,vector constructs in which the antisense nucleic acid molecule is placedunder the control of a strong pol II or pol III promoter are preferred.

An antisense nucleic acid molecule of the invention can be an α-anomericnucleic acid molecule. An α-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

The invention also encompasses ribozymes, which are catalytic RNAmolecules with ribonuclease activity that are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Ribozymes (e.g., hammerhead ribozymes (describedin Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used tocatalytically cleave human phospholipid scramblase-like mRNA transcriptsto thereby inhibit translation of human phospholipid scramblase-likemRNA. A ribozyme having specificity for a human phospholipidscramblase-like-encoding nucleic acid can be designed based upon thenucleotide sequence of a human phospholipid scramblase-like cDNAdisclosed herein (e.g., SEQ ID NO:17). See, e.g., Cech et al., U.S. Pat.No. 4,987,071; and Cech et al., U.S. Pat. No. 5,116,742. Alternatively,human phospholipid scramblase-like mRNA can be used to select acatalytic RNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel and Szostak (1993) Science 261:1411-1418.

The invention also encompasses nucleic acid molecules that form triplehelical structures. For example, human phospholipid scramblase-like geneexpression can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the human phospholipidscramblase-like protein (e.g., the human phospholipid scramblase-likepromoter and/or enhancers) to form triple helical structures thatprevent transcription of the human phospholipid scramblase-like gene intarget cells. See generally Helene (1991) Anticancer Drug Des. 6(6):569;Helene (1992) Ann. N.Y. Acad. Sci. 660:27; and Maher (1992) Bioassays14(12):807.

In preferred embodiments, the nucleic acid molecules of the inventioncan be modified at the base moiety, sugar moiety, or phosphate backboneto improve, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal. (1996) Bioorganic & Medicinal Chemistry 4:5). As used herein, theterms “peptide nucleic acids” or “PNAs” refer to nucleic acid mirnics,e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid-phase peptide synthesis protocols as described in Hyrupet al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci.USA 93:14670.

PNAs of a human phospholipid scramblase-like molecule can be used intherapeutic and diagnostic applications. For example, PNAs can be usedas antisense or antigene agents for sequence-specific modulation of geneexpression by, e.g., inducing transcription or translation arrest orinhibiting replication. PNAs of the invention can also be used, e.g., inthe analysis of single base pair mutations in a gene by, e.g.,PNA-directed PCR clamping; as artificial restriction enzymes when usedin combination with other enzymes, e.g., S1 nucleases (Hyrup (1996),supra; or as probes or primers for DNA sequence and hybridization (Hyrup(1996), supra; Perry-O'Keefe et al. (1996), supra).

In another embodiment, PNAs of a human phospholipid scramblase-likemolecule can be modified, e.g., to enhance their stability, specificity,or cellular uptake, by attaching lipophilic or other helper groups toPNA, by the formation of PNA-DNA chimeras, or by the use of liposomes orother techniques of drug delivery known in the art. The synthesis ofPNA-DNA chimeras can be performed as described in Hyrup (1996), supra;Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63; Mag et al. (1989)Nucleic Acids Res. 17:5973; and Peterson et al. (1975) Bioorganic Med.Chem. Lett. 5:1119.

II. Isolated Human Phospholipid Scramblase-Like Proteins and Anti-HumanPhospholipid Scramblase-Like Antibodies

Human phospholipid scramblase-like proteins are also encompassed withinthe present invention. By “human phospholipid scramblase-like protein”is intended a protein having the amino acid sequence set forth in SEQ IDNO:17, as well as fragments, biologically active portions, and variantsthereof.

“Fragments” or “biologically active portions” include polypeptidefragments suitable for use as immunogens to raise anti-humanphospholipid scramblase-like antibodies. Fragments include peptidescomprising amino acid sequences sufficiently identical to or derivedfrom the amino acid sequence of a human phospholipid scramblase-likeprotein, or partial-length protein, of the invention and exhibiting atleast one activity of a human phospholipid scramblase-like protein, butwhich include fewer amino acids than the full-length (SEQ ID NO:17)human phospholipid scramblase-like protein disclosed herein. Typically,biologically active portions comprise a domain or motif with at leastone activity of the human phospholipid scramblase-like protein. Abiologically active portion of a human phospholipid scramblase-likeprotein can be a polypeptide which is, for example, 10, 25, 50, 100 ormore amino acids in length. Such biologically active portions can beprepared by recombinant techniques and evaluated for one or more of thefunctional activities of a native human phospholipid scramblase-likeprotein. As used here, a fragment comprises at least 5 contiguous aminoacids of SEQ ID NO:17. The invention encompasses other fragments,however, such as any fragment in the protein greater than 6, 7, 8, or 9amino acids.

By “variants” is intended proteins or polypeptides having an amino acidsequence that is at least about 60%, 65%, or 70%, preferably about 75%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of SEQ ID NO:17. Variants also include polypeptidesencoded by a nucleic acid molecule that hybridizes to the nucleic acidmolecules of SEQ ID NO:16, SEQ ID NO:18, or a complement thereof, understringent conditions. In another embodiment, a variant of an isolatedpolypeptide of the present invention differs, by at least 1, but lessthan 5, 10, 20, 50, or 100 amino acid residues from the sequence shownin SEQ ID NO:17. If alignment is needed for this comparison thesequences should be aligned for maximum identity. “Looped” out sequencesfrom deletions or insertions, or mismatches, are considered differences.Such variants generally retain the functional activity of the 32621-likeproteins of the invention. Variants include polypeptides that differ inamino acid sequence due to natural allelic variation or mutagenesis.

In one embodiment, a 32621-like protein includes at least onetransmembrane domain. As used herein, the term “transmembrane domain”includes an amino acid sequence of about 15 amino acid residues inlength that spans a phospholipid membrane. More preferably, atransmembrane domain includes about at least 16, 18, or 20 amino acidresidues and spans a phospholipid membrane. Transmembrane domains arerich in hydrophobic residues, and typically have an α-helical structure.In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or moreof the amino acids of a transmembrane domain are hydrophobic, e.g.,leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domainsare described in, for example,http://pfam.wustl.edu/cgi-bin/getdesc?name=7tm-1, and Zagotta W. N. etal. (1996) Annual Rev. Neuronsci. 19:235-63, the contents of which areincorporated herein by reference.

In a preferred embodiment, a 32621-like polypeptide or protein has atleast one transmembrane domain or a region which includes at least 15,16, 18, or 20 amino acid residues and has at least about 60%, 70% 80%90% 95%, 99%, or 100% sequence identity with a “transmembrane domain,”e.g., at least one transmembrane domain of human 32621-like (e.g., aminoacid residues 304-320 of SEQ ID NO:17).

In another embodiment, a 32621-like protein includes at least one“non-transmembrane domain.” As used herein, “non-transmembrane domains”are domains that reside outside of the membrane. When referring toplasma membranes, non-transmembrane domains include extracellulardomains (i.e., outside of the cell) and intracellular domains (i.e.,within the cell). When referring to membrane-bound proteins found inintracellular organelles (e.g., mitochondria, endoplasmic reticulum,peroxisomes and microsomes), non-transmembrane domains include thosedomains of the protein that reside in the cytosol (i.e., the cytoplasm),the lumen of the organelle, or the matrix or the intermembrane space(the latter two relate specifically to mitochondria organelles). TheC-terminal amino acid residue of a non-transmembrane domain is adjacentto an N-terminal amino acid residue of a transmembrane domain in anaturally occurring 32621-like, or 32621 like protein.

In a preferred embodiment, a 32621-like polypeptide or protein has a“non-transmembrane domain” or a region which includes at least about1-312, preferably about 200-312, more preferably about 230-300, and evenmore preferably about 240-280 amino acid residues, and has at leastabout 60%, 70% 80% 90% 95%, 99% or 100% sequence identity with a“non-transmembrane domain”, e.g., a non-transmembrane domain of human32621-like (e.g., residues 1-303 or 321-329 of SEQ ID NO:17).Preferably, a non-transmembrane domain is capable of catalytic activity(e.g., phospholipid scramblase activity).

A non-transmembrane domain located at the N-terminus of a 32621-likeprotein or polypeptide is referred to herein as an “N-terminalnon-transmembrane domain.” As used herein, an “N-terminalnon-transmembrane domain” includes an amino acid sequence having about1-303, preferably about 30-303, more preferably about 50-303, or evenmore preferably about 80-290 amino acid residues in length and islocated outside the boundaries of a membrane. For example, an N-terminalnon-transmembrane domain is located at about amino acid residues 1-303of SEQ ID NO:17.

Similarly, a non-transmembrane domain located at the C-terminus of a32621-like protein or polypeptide is referred to herein as a “C-terminalnon-transmembrane domain.” As used herein, an “C-terminalnon-transmembrane domain” includes an amino acid sequence having about1-300, preferably about 15-290, preferably about 20-270, more preferablyabout 25-255 amino acid residues in length and is located outside theboundaries of a membrane. For example, an C-terminal non-transmembranedomain is located at about amino acid residues 321-329 of SEQ ID NO:17.

The invention also provides human phospholipid scramblase-like chimericor fusion proteins. As used herein, a human phospholipid scramblase-like“chimeric protein” or “fusion protein” comprises a human phospholipidscramblase-like polypeptide operably linked to a non-human phospholipidscramblase-like polypeptide. A “human phospholipid scramblase-likepolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to a human phospholipid scramblase-like protein, whereas a“non-human phospholipid scramblase-like polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a proteinthat is not substantially identical to the human phospholipidscramblase-like protein, e.g., a protein that is different from thehuman phospholipid scramblase-like protein and which is derived from thesame or a different organism. Within a human phospholipidscramblase-like fusion protein, the human phospholipid scramblase-likepolypeptide can correspond to all or a portion of a human phospholipidscramblase-like protein, preferably at least one biologically activeportion of a human phospholipid scramblase-like protein. Within thefusion protein, the term “operably linked” is intended to indicate thatthe human phospholipid scramblase-like polypeptide and the non-humanphospholipid scramblase-like polypeptide are fused in-frame to eachother. The non-human phospholipid scramblase-like polypeptide can befused to the N-terminus or C-terminus of the human phospholipidscramblase-like polypeptide.

One useful fusion protein is a GST-human phospholipid scramblase-likefusion protein in which the human phospholipid scramblase-like sequencesare fused to the C-terminus of the GST sequences. Such fusion proteinscan facilitate the purification of recombinant human phospholipidscramblase-like proteins.

In yet another embodiment, the fusion protein is a human phospholipidscramblase-like-immunoglobulin fusion protein in which all or part of ahuman phospholipid scramblase-like protein is fused to sequences derivedfrom a member of the immunoglobulin protein family. The humanphospholipid scramblase-like-immunoglobulin fusion proteins of theinvention can be incorporated into pharmaceutical compositions andadministered to a subject to inhibit an interaction between a humanphospholipid scramblase-like ligand and a human phospholipidscramblase-like protein on the surface of a cell, thereby suppressinghuman phospholipid scramblase-like-mediated signal transduction in vivo.The human phospholipid scramblase-like-immunoglobulin fusion proteinscan be used to affect the bioavailability of a human phospholipidscramblase-like cognate ligand. Inhibition of the human phospholipidscramblase-like ligand/human phospholipid scramblase-like interactionmay be useful therapeutically. Moreover, the human phospholipidscramblase-like-immunoglobulin fusion proteins of the invention can beused as immunogens to produce anti-human phospholipid scramblase-likeantibodies in a subject, to purify human phospholipid scramblase-likeligands, and in screening assays to identify molecules that inhibit theinteraction of a human phospholipid scramblase-like protein with a humanphospholipid scramblase-like ligand.

Preferably, a human phospholipid scramblase-like chimeric or fusionprotein of the invention is produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the differentpolypeptide sequences may be ligated together in-frame, or the fusiongene can be synthesized, such as with automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers that give rise to complementary overhangs betweentwo consecutive gene fragments, which can subsequently be annealed andreamplified to generate a chimeric gene sequence (see, e.g., Ausubel etal., eds. (1995) Current Protocols in Molecular Biology) (GreenePublishing and Wiley-Interscience, NY). Moreover, a human phospholipidscramblase-like-encoding nucleic acid can be cloned into a commerciallyavailable expression vector such that it is linked in-frame to anexisting fusion moiety.

Variants of the human phospholipid scramblase-like proteins can functionas either human phospholipid scramblase-like agonists (mimetics) or ashuman phospholipid scramblase-like antagonists. Variants of the humanphospholipid scramblase-like protein can be generated by mutagenesis,e.g., discrete point mutation or truncation of the human phospholipidscramblase-like protein. An agonist of the human phospholipidscramblase-like protein can retain substantially the same, or a subset,of the biological activities of the naturally occurring form of thehuman phospholipid scramblase-like protein. An antagonist of the humanphospholipid scramblase-like protein can inhibit one or more of theactivities of the naturally occurring form of the human phospholipidscramblase-like protein by, for example, competitively binding to adownstream or upstream member of a cellular signaling cascade thatincludes the human phospholipid scramblase-like protein. Thus, specificbiological effects can be elicited by treatment with a variant oflimited function. Treatment of a subject with a variant having a subsetof the biological activities of the naturally occurring form of theprotein can have fewer side effects in a subject relative to treatmentwith the naturally occurring form of the human phospholipidscramblase-like proteins.

Variants of a human phospholipid scramblase-like protein that functionas either human phospholipid scramblase-like agonists or as humanphospholipid scramblase-like antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of a humanphospholipid scramblase-like protein for human phospholipidscramblase-like protein agonist or antagonist activity. In oneembodiment, a variegated library of human phospholipid scramblase-likevariants is generated by combinatorial mutagenesis at the nucleic acidlevel and is encoded by a variegated gene library. A variegated libraryof human phospholipid scramblase-like variants can be produced by, forexample, enzymatically ligating a mixture of synthetic oligonucleotidesinto gene sequences such that a degenerate set of potential humanphospholipid scramblase-like sequences is expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display) containing the set of human phospholipidscramblase-like sequences therein. There are a variety of methods thatcan be used to produce libraries of potential human phospholipidscramblase-like variants from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be performed in anautomatic DNA synthesizer, and the synthetic gene then ligated into anappropriate expression vector. Use of a degenerate set of genes allowsfor the provision, in one mixture, of all of the sequences encoding thedesired set of potential human phospholipid scramblase-like sequences.Methods for synthesizing degenerate oligonucleotides are known in theart (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984)Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ikeet al. (1983) Nucleic Acid Res. 11:477).

In addition, libraries of fragments of a human phospholipidscramblase-like protein coding sequence can be used to generate avariegated population of human phospholipid scramblase-like fragmentsfor screening and subsequent selection of variants of a humanphospholipid scramblase-like protein. In one embodiment, a library ofcoding sequence fragments can be generated by treating a double-strandedPCR fragment of a human phospholipid scramblase-like coding sequencewith a nuclease under conditions wherein nicking occurs only about onceper molecule, denaturing the double-stranded DNA, renaturing the DNA toform double-stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single-stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method,one can derive an expression library that encodes N-terminal andinternal fragments of various sizes of the human phospholipidscramblase-like protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of human phospholipidscramblase-like proteins. The most widely used techniques, which areamenable to high through-put analysis, for screening large genelibraries typically include cloning the gene library into replicableexpression vectors, transforming appropriate cells with the resultinglibrary of vectors, and expressing the combinatorial genes underconditions in which detection of a desired activity facilitatesisolation of the vector encoding the gene whose product was detected.Recursive ensemble mutagenesis (REM), a technique that enhances thefrequency of functional mutants in the libraries, can be used incombination with the screening assays to identify human phospholipidscramblase-like variants (Arkin and Yourvan (1992) Proc. Natl. Acad.Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering6(3):327-331).

An isolated human phospholipid scramblase-like polypeptide of theinvention can be used as an immunogen to generate antibodies that bindhuman phospholipid scramblase-like proteins using standard techniquesfor polyclonal and monoclonal antibody preparation. The full-lengthhuman phospholipid scramblase-like protein can be used or,alternatively, the invention provides antigenic peptide fragments ofhuman phospholipid scramblase-like proteins for use as immunogens. Theantigenic peptide of a human phospholipid scramblase-like proteincomprises at least 8, preferably 10, 15, 20, or 30 amino acid residuesof the amino acid sequence shown in SEQ ID NO:17 and encompasses anepitope of a human phospholipid scramblase-like protein such that anantibody raised against the peptide forms a specific immune complex withthe human phospholipid scramblase-like protein. Preferred epitopesencompassed by the antigenic peptide are regions of a human phospholipidscramblase-like protein that are located on the surface of the protein,e.g., hydrophilic regions.

Accordingly, another aspect of the invention pertains to anti-humanphospholipid scramblase-like polyclonal and monoclonal antibodies thatbind a human phospholipid scramblase-like protein. Polyclonal anti-humanphospholipid scramblase-like antibodies can be prepared by immunizing asuitable subject (e.g., rabbit, goat, mouse, or other mammal) with ahuman phospholipid scramblase-like immunogen. The anti-humanphospholipid scramblase-like antibody titer in the immunized subject canbe monitored over time by standard techniques, such as with an enzymelinked immunosorbent assay (ELISA) using immobilized human phospholipidscramblase-like protein. At an appropriate time after immunization,e.g., when the anti-human phospholipid scramblase-like antibody titersare highest, antibody-producing cells can be obtained from the subjectand used to prepare monoclonal antibodies by standard techniques, suchas the hybridoma technique originally described by Kohler and Milstein(1975) Nature 256:495-497, the human B cell hybridoma technique (Kozboret al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole etal. (1985) in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld andSell (Alan R. Liss, Inc., New York, N.Y.), pp. 77-96) or triomatechniques. The technology for producing hybridomas is well known (seegenerally Coligan et al., eds. (1994) Current Protocols in Immunology(John Wiley & Sons, Inc., New York, N.Y.); Galfre et al. (1977) Nature266:550-52; Kenneth (1980) in Monoclonal Antibodies: A New Dimension InBiological Analyses (Plenum Publishing Corp., NY; and Lerner (1981) YaleJ. Biol. Med., 54:387-402).

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-human phospholipid scramblase-like antibody can beidentified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) with ahuman phospholipid scramblase-like protein to thereby isolateimmunoglobulin library members that bind the human phospholipidscramblase-like protein. Kits for generating and screening phage displaylibraries are commercially available (e.g., the Pharmacia RecombinantPhage Antibody System, Catalog No. 27-9400-01; and the StratageneSurfZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examplesof methods and reagents particularly amenable for use in generating andscreening antibody display library can be found in, for example, U.S.Pat. No. 5,223,409; PCT Publication Nos. WO 92/18619; WO 91/17271; WO92/20791; WO 92/15679; 93/01288; WO 92/01047; 92/09690; and 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffiths et al. (1993) EMBO J. 12:725-734.

Additionally, recombinant anti-human phospholipid scramblase-likeantibodies, such as chimeric and humanized monoclonal antibodies,comprising both human and nonhuman portions, which can be made usingstandard recombinant DNA techniques, are within the scope of theinvention. Such chimeric and humanized monoclonal antibodies can beproduced by recombinant DNA techniques known in the art, for exampleusing methods described in PCT Publication Nos. WO 86/01533 and WO87/02671; European Patent Application Nos. 184,187, 171, 496, 125,023,and 173,494; U.S. Pat. Nos. 4,816,567 and 5,225,539; European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986)Bio/Techniques 4:214; Jones et al. (1986) Nature 321:552-525; Verhoeyanet al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.141:4053-4060.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice that are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. See for example, Lonberg and Huszar (1995)Int. Rev. Immunol. 13:65-93); and U.S. Pat. Nos. 5,625,126; 5,633,425;5,569,825; 5,661,016; and 5,545,806. In addition, companies such asAbgenix, Inc. (Freemont, Calif.), can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. This technology is described by Jespers etal. (1994) Bio/Technology 12:899-903).

An anti-human phospholipid scramblase-like antibody (e.g., monoclonalantibody) can be used to isolate human phospholipid scramblase-likeproteins by standard techniques, such as affinity chromatography orimmunoprecipitation. An anti-human phospholipid scramblase-like antibodycan facilitate the purification of natural human phospholipidscramblase-like protein from cells and of recombinantly produced humanphospholipid scramblase-like protein expressed in host cells. Moreover,an anti-human phospholipid scramblase-like antibody can be used todetect human phospholipid scramblase-like protein (e.g., in a cellularlysate or cell supernatant) in order to evaluate the abundance andpattern of expression of the human phospholipid scramblase-like protein.Anti-human phospholipid scramblase-like antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling theantibody to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S,or ³H.

Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). The conjugates of the invention canbe used for modifying a given biological response, the drug moiety isnot to be construed as limited to classical chemical therapeutic agents.For example, the drug moiety may be a protein or polypeptide possessinga desired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, alpha-interferon,beta-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies'84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a humanphospholipid scramblase-like protein (or a portion thereof). “Vector”refers to a nucleic acid molecule capable of transporting anothernucleic acid to which it has been linked, such as a “plasmid”, acircular double-stranded DNA loop into which additional DNA segments canbe ligated, or a viral vector, where additional DNA segments can beligated into the viral genome. The vectors are useful for autonomousreplication in a host cell or may be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome (e.g., nonepisomal mammalianvectors). Expression vectors are capable of directing the expression ofgenes to which they are operably linked. In general, expression vectorsof utility in recombinant DNA techniques are often in the form ofplasmids (vectors). However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses, and adeno-associatedviruses), that serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell. This means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, operably linked to the nucleicacid sequence to be expressed. “Operably linked” is intended to meanthat the nucleotide sequence of interest is linked to the regulatorysequence(s) in a manner that allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in ahost cell when the vector is introduced into the host cell). The term“regulatory sequence” is intended to include promoters, enhancers, andother expression control elements (e.g., polyadenylation signals). See,for example, Goeddel (1990) in Gene Expression Technology: Methods inEnzymology 185 (Academic Press, San Diego, Calif.). Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cell and those that direct expression ofthe nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., human phospholipidscramblase-like proteins, mutant forms of human phospholipidscramblase-like proteins, fusion proteins, etc.). It is furtherrecognized that the nucleic acid sequences of the invention can bealtered to contain codons, which are preferred, or non preferred, for aparticular expression system. For example, the nucleic acid can be onein which at least one altered codon, and preferably at least 10%, or 20%of the codons have been altered such that the sequence is optimized forexpression in E. coli, yeast, human, insect, or CHO cells. Methods fordetermining such codon usage are well known in the art.

The recombinant expression vectors of the invention can be designed forexpression of human phospholipid scramblase-like protein in prokaryoticor eukaryotic host cells. Expression of proteins in prokaryotes is mostoften carried out in E. coli with vectors containing constitutive orinducible promoters directing the expression of either fusion ornonfusion proteins. Fusion vectors add a number of amino acids to aprotein encoded therein, usually to the amino terminus of therecombinant protein. Typical fusion expression vectors include pGEX(Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40), pMAL(New England Biolabs, Beverly, Mass.), and pRIT5 (Pharmacia, Piscataway,N.J.) which fuse glutathione S-transferase (GST), maltose E bindingprotein, or protein A, respectively, to the target recombinant protein.Examples of suitable inducible nonfusion E. coli expression vectorsinclude pTrc (Amann et al. (1988) Gene 69:301-315) and pET 11d (Studieret al. (1990) in Gene Expression Technology: Methods in Enzymology 185(Academic Press, San Diego, Calif.), pp. 60-89). Strategies to maximizerecombinant protein expression in E. coli can be found in Gottesman(1990) in Gene Expression Technology: Methods in Enzymology 185(Academic Press, CA), pp. 119-128 and Wada et al. (1992) Nucleic AcidsRes. 20:2111-2118. Target gene expression from the pTrc vector relies onhost RNA polymerase transcription from a hybrid trp-lac fusion promoter.

Suitable eukaryotic host cells include insect cells (examples ofBaculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39)); yeast cells (examples of vectorsfor expression in yeast S. cerevisiae include pYepSec1 (Baldari et al.(1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2(Invitrogen Corporation, San Diego, Calif.), and pPicZ (InvitrogenCorporation, San Diego, Calif.)); or mammalian cells (mammalianexpression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC(Kaufman et al. (1987) EMBO J. 6:187:195)). Suitable mammalian cellsinclude Chinese hamster ovary cells (CHO) or COS cells. In mammaliancells, the expression vector's control functions are often provided byviral regulatory elements. For example, commonly used promoters arederived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus40. For other suitable expression systems for both prokaryotic andeukaryotic cells, see chapters 16 and 17 of Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.). See Goeddel (1990) in GeneExpression Technology: Methods in Enzymology 185 (Academic Press, SanDiego, Calif.). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but also to the progeny or potentialprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell but are still included within the scope of the term as used herein.A “purified preparation of cells”, as used herein, refers to, in thecase of plant or animal cells, an in vitro preparation of cells and notan entire intact plant or animal. In the case of cultured cells ormicrobial cells, it consists of a preparation of at least 10% and morepreferably 50% of the subject cells.

In one embodiment, the expression vector is a recombinant mammalianexpression vector that comprises tissue-specific regulatory elementsthat direct expression of the nucleic acid preferentially in aparticular cell type. Suitable tissue-specific promoters include thealbumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev.1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv.Immunol. 43:235-275), in particular promoters of T cell receptors(Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins(Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell33:741-748), neuron-specific promoters (e.g., the neurofilamentpromoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science230:912-916), and mammary gland-specific promoters (e.g., milk wheypromoter; U.S. Pat. No. 4,873,316 and European Application PatentPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379), the a-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546), and the like.

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperably linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to human phospholipid scramblase-like mRNA. Regulatorysequences operably linked to a nucleic acid cloned in the antisenseorientation can be chosen to direct the continuous expression of theantisense RNA molecule in a variety of cell types, for instance viralpromoters and/or enhancers, or regulatory sequences can be chosen todirect constitutive, tissue-specific, or cell-type-specific expressionof antisense RNA. The antisense expression vector can be in the form ofa recombinant plasmid, phagemid, or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub et al.(1986) Reviews—Trends in Genetics, Vol. 1(1).

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAF-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.) and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those that confer resistance todrugs, such as G418, hygromycin, and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding a human phospholipid scramblase-like protein orcan be introduced on a separate vector. Cells stably transfected withthe introduced nucleic acid can be identified by drug selection (e.g.,cells that have incorporated the selectable marker gene will survive,while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) humanphospholipid scramblase-like protein. Accordingly, the invention furtherprovides methods for producing human phospholipid scramblase-likeprotein using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of the invention, into which arecombinant expression vector encoding a human phospholipidscramblase-like protein has been introduced, in a suitable medium suchthat human phospholipid scramblase-like protein is produced. In anotherembodiment, the method further comprises isolating human phospholipidscramblase-like protein from the medium or the host cell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichhuman phospholipid scramblase-like-coding sequences have beenintroduced. Such host cells can then be used to create nonhumantransgenic animals in which exogenous human phospholipid scramblase-likesequences have been introduced into their genome or homologousrecombinant animals in which endogenous human phospholipidscramblase-like sequences have been altered. Such animals are useful forstudying the function and/or activity of human phospholipidscramblase-like genes and proteins and for identifying and/or evaluatingmodulators of human phospholipid scramblase-like activity. As usedherein, a “transgenic animal” is a nonhuman animal, preferably a mammal,more preferably a rodent such as a rat or mouse, in which one or more ofthe cells of the animal includes a transgene. Other examples oftransgenic animals include nonhuman primates, sheep, dogs, cows, goats,chickens, amphibians, etc. A transgene is exogenous DNA that isintegrated into the genome of a cell from which a transgenic animaldevelops and which remains in the genome of the mature animal, therebydirecting the expression of an encoded gene product in one or more celltypes or tissues of the transgenic animal. As used herein, a “homologousrecombinant animal” is a nonhuman animal, preferably a mammal, morepreferably a mouse, in which an endogenous human phospholipidscramblase-like gene has been altered by homologous recombinationbetween the endogenous gene and an exogenous DNA molecule introducedinto a cell of the animal, e.g., an embryonic cell of the animal, priorto development of the animal.

A transgenic animal of the invention can be created by introducing humanphospholipid scramblase-like-encoding nucleic acid into the malepronuclei of a fertilized oocyte, e.g., by microinjection, retroviralinfection, and allowing the oocyte to develop in a pseudopregnant femalefoster animal. The human phospholipid scramblase-like cDNA sequence canbe introduced as a transgene into the genome of a nonhuman animal.Alternatively, a homologue of the mouse human phospholipidscramblase-like gene can be isolated based on hybridization and used asa transgene. Intronic sequences and polyadenylation signals can also beincluded in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operablylinked to the human phospholipid scramblase-like transgene to directexpression of human phospholipid scramblase-like protein to particularcells. Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866, 4,870,009, and 4,873,191 and in Hogan (1986)Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1986). Similar methods are used for production ofother transgenic animals. A transgenic founder animal can be identifiedbased upon the presence of the human phospholipid scramblase-liketransgene in its genome and/or expression of human phospholipidscramblase-like mRNA in tissues or cells of the animals. A transgenicfounder animal can then be used to breed additional animals carrying thetransgene. Moreover, transgenic animals carrying a transgene encodinghuman phospholipid scramblase-like gene can further be bred to othertransgenic animals carrying other transgenes.

To create a homologous recombinant animal, one prepares a vectorcontaining at least a portion of a human phospholipid scramblase-likegene or a homolog of the gene into which a deletion, addition, orsubstitution has been introduced to thereby alter, e.g., functionallydisrupt, the human phospholipid scramblase-like gene. In a preferredembodiment, the vector is designed such that, upon homologousrecombination, the endogenous human phospholipid scramblase-like gene isfunctionally disrupted (i.e., no longer encodes a functional protein;also referred to as a “knock out” vector). Alternatively, the vector canbe designed such that, upon homologous recombination, the endogenoushuman phospholipid scramblase-like gene is mutated or otherwise alteredbut still encodes functional protein (e.g., the upstream regulatoryregion can be altered to thereby alter the expression of the endogenoushuman phospholipid scramblase-like protein). In the homologousrecombination vector, the altered portion of the human phospholipidscramblase-like gene is flanked at its 5′ and 3′ ends by additionalnucleic acid of the human phospholipid scramblase-like gene to allow forhomologous recombination to occur between the exogenous humanphospholipid scramblase-like gene carried by the vector and anendogenous human phospholipid scramblase-like gene in an embryonic stemcell. The additional flanking human phospholipid scramblase-like nucleicacid is of sufficient length for successful homologous recombinationwith the endogenous gene. Typically, several kilobases of flanking DNA(both at the 5′ and 3′ ends) are included in the vector (see, e.g.,Thomas and Capecchi (1987) Cell 51:503 for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation), and cells in which the introducedhuman phospholipid scramblase-like gene has homologously recombined withthe endogenous human phospholipid scramblase-like gene are selected(see, e.g., Li et al. (1992) Cell 69:915). The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse) to formaggregation chimeras (see, e.g., Bradley (1987) in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, ed. Robertson (IRL, Oxfordpp. 113-152). A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term.Progeny harboring the homologously recombined DNA in their germ cellscan be used to breed animals in which all cells of the animal containthe homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination vectors andhomologous recombinant animals are described further in Bradley (1991)Current Opinion in Bio/Techniques 2:823-829 and in PCT Publication Nos.WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169.

In another embodiment, transgenic nonhuman animals containing selectedsystems that allow for regulated expression of the transgene can beproduced. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355). If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the nonhuman transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and PCT Publication Nos. WO 97/07668 and WO 97/07669.

IV. Pharmaceutical Compositions

The human phospholipid scramblase-like nucleic acid molecules, humanphospholipid scramblase-like proteins, and anti-human phospholipidscramblase-like antibodies (also referred to herein as “activecompounds”) of the invention can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise the nucleic acid molecule, protein, or antibody and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The compositions of the invention are useful to treat any of thedisorders discussed herein. The compositions are provided intherapeutically effective amounts. By “therapeutically effectiveamounts” is intended an amount sufficient to modulate the desiredresponse. As defined herein, a therapeutically effective amount ofprotein or polypeptide (i.e., an effective dosage) ranges from about0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg bodyweight, more preferably about 0.1 to 20 mg/kg body weight, and even morepreferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7mg/kg, or 5 to 6 mg/kg body weight.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of a protein, polypeptide, or antibody can include asingle treatment or, preferably, can include a series of treatments. Ina preferred example, a subject is treated with antibody, protein, orpolypeptide in the range of between about 0.1 to 20 mg/kg body weight,one time per week for between about 1 to 10 weeks, preferably between 2to 8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody, protein, or polypeptide used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays as described herein.

The present invention encompasses agents which modulate expression oractivity. An agent may, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e., including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds.

It is understood that appropriate doses of small molecule agents dependsupon a number of factors within the skill of the ordinarily skilledphysician, veterinarian, or researcher. The dose(s) of the smallmolecule will vary, for example, depending upon the identity, size, andcondition of the subject or sample being treated, further depending uponthe route by which the composition is to be administered, if applicable,and the effect which the practitioner desires the small molecule to haveupon the nucleic acid or polypeptide of the invention. Exemplary dosesinclude milligram or microgram amounts of the small molecule perkilogram of subject or sample weight (e.g., about 1 microgram perkilogram to about 500 milligrams per kilogram, about 100 micrograms perkilogram to about 5 milligrams per kilogram, or about 1 microgram perkilogram to about 50 micrograms per kilogram. It is furthermoreunderstood that appropriate doses of a small molecule depend upon thepotency of the small molecule with respect to the expression or activityto be modulated. Such appropriate doses may be determined using theassays described herein. When one or more of these small molecules is tobe administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerin, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes, or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF; Parsippany, N.J.), or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion, and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride, inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a human phospholipid scramblase-like protein oranti-human phospholipid scramblase-like antibody) in the required amountin an appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying,which yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth, or gelatin; an excipientsuch as starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. For administrationby inhalation, the compounds are delivered in the form of an aerosolspray from a pressurized container or dispenser that contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art. Thecompounds can also be prepared in the form of suppositories (e.g., withconventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated with each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. Depending on thetype and severity of the disease, about 1 μg/kg to about 15 mg/kg (e.g.,0.1 to 20 mg/kg) of antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to about 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays. Anexemplary dosing regimen is disclosed in WO 94/04188. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470), or by stereotactic injection(see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods:(a) screening assays; (b) detection assays (e.g., chromosomal mapping,tissue typing, forensic biology); (c) predictive medicine (e.g.,diagnostic assays, prognostic assays, monitoring clinical trials, andpharmacogenomics); and (d) methods of treatment (e.g., therapeutic andprophylactic). The isolated nucleic acid molecules of the invention canbe used to express human phospholipid scramblase-like protein (e.g., viaa recombinant expression vector in a host cell in gene therapyapplications), to detect human phospholipid scramblase-like mRNA (e.g.,in a biological sample) or a genetic lesion in a human phospholipidscramblase-like gene, and to modulate human phospholipid scramblase-likeactivity. In addition, the human phospholipid scramblase-like proteinscan be used to screen drugs or compounds that modulate the immune,hemopoetic, and blood clotting responses as well as to treat disorderscharacterized by insufficient or excessive production of humanphospholipid scramblase-like protein or production of human phospholipidscramblase-like protein forms that have decreased or aberrant activitycompared to human phospholipid scramblase-like wild type protein. Inaddition, the anti-human phospholipid scramblase-like antibodies of theinvention can be used to detect and isolate human phospholipidscramblase-like proteins and modulate human phospholipid scramblase-likeactivity.

A. Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules, or otherdrugs) that bind to human phospholipid scramblase-like proteins or havea stimulatory or inhibitory effect on, for example, human phospholipidscramblase-like expression or human phospholipid scramblase-likeactivity.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including biological libraries, spatially addressable parallelsolid phase or solution phase libraries, synthetic library methodsrequiring deconvolution, the “one-bead one-compound” library method, andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, nonpeptide oligomer, orsmall molecule libraries of compounds (Lam (1997) Anticancer Drug Des.12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andGallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Bio/Techniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Pat.No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA89:1865-1869), or phage (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. USA 87:6378-6382; and Felici (1991) J. Mol. Biol.222:301-310).

Determining the ability of the test compound to bind to the humanphospholipid scramblase-like protein can be accomplished, for example,by coupling the test compound with a radioisotope or enzymatic labelsuch that binding of the test compound to the human phospholipidscramblase-like protein or biologically active portion thereof can bedetermined by detecting the labeled compound in a complex. For example,test compounds can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, eitherdirectly or indirectly, and the radioisotope detected by direct countingof radioemmission or by scintillation counting. Alternatively, testcompounds can be enzymatically labeled with, for example, horseradishperoxidase, alkaline phosphatase, or luciferase, and the enzymatic labeldetected by determination of conversion of an appropriate substrate toproduct.

In a similar manner, one may determine the ability of the humanphospholipid scramblase-like protein to bind to or interact with a humanphospholipid scramblase-like target molecule. By “target molecule” isintended a molecule with which a human phospholipid scramblase-likeprotein binds or interacts in nature. In a preferred embodiment, theability of the human phospholipid scramblase-like protein to bind to orinteract with a human phospholipid scramblase-like target molecule(s)can be determined by monitoring the activity of the target molecule. Forexample, the activity of the PS scramblase can be monitored by detectingthe translocation of phospholipids in the plasma membrane in response toelevated Ca⁺² (Zhao, J. et al. (1998) J. Biol. Chem. 273(12): 6603-6606.

In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting a human phospholipidscramblase-like protein or biologically active portion thereof with atest compound and determining the ability of the test compound to bindto the human phospholipid scramblase-like protein or biologically activeportion thereof. Binding of the test compound to the human phospholipidscramblase-like protein can be determined either directly or indirectlyas described above. In a preferred embodiment, the assay includescontacting the human phospholipid scramblase-like protein orbiologically active portion thereof with a known compound that bindshuman phospholipid scramblase-like protein to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to preferentially bind to humanphospholipid scramblase-like protein or biologically active portionthereof as compared to the known compound.

In another embodiment, an assay is a cell-free assay comprisingcontacting human phospholipid scramblase-like protein or biologicallyactive portion thereof with a test compound and determining the abilityof the test compound to modulate (e.g., stimulate or inhibit) theactivity of the human phospholipid scramblase-like protein orbiologically active portion thereof. Determining the ability of the testcompound to modulate the activity of a human phospholipidscramblase-like protein can be accomplished, for example, by determiningthe ability of the human phospholipid scramblase-like protein to bind toa human phospholipid scramblase-like target molecule as described abovefor determining direct binding. In an alternative embodiment,determining the ability of the test compound to modulate the activity ofa human phospholipid scramblase-like protein can be accomplished bydetermining the ability of the human phospholipid scramblase-likeprotein to further modulate a human phospholipid scramblase-like targetmolecule. For example, the catalytic/enzymatic activity of the targetmolecule on an appropriate substrate can be determined as previouslydescribed.

In yet another embodiment, the cell-free assay comprises contacting thehuman phospholipid scramblase-like protein or biologically activeportion thereof with a known compound that binds a human phospholipidscramblase-like protein to form an assay mixture, contacting the assaymixture with a test compound, and determining the ability of the testcompound to preferentially bind to or modulate the activity of a humanphospholipid scramblase-like target molecule.

In the above-mentioned assays, it may be desirable to immobilize eithera human phospholipid scramblase-like protein or its target molecule tofacilitate separation of complexed from uncomplexed forms of one or bothof the proteins, as well as to accommodate automation of the assay. Inone embodiment, a fusion protein can be provided that adds a domain thatallows one or both of the proteins to be bound to a matrix. For example,glutathione-S-transferase/human phospholipid scramblase-like fusionproteins or glutathione-S-transferase/target fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione-derivatized microtitre plates, which are thencombined with the test compound or the test compound and either thenonadsorbed target protein or human phospholipid scramblase-likeprotein, and the mixture incubated under conditions conducive to complexformation (e.g., at physiological conditions for salt and pH). Followingincubation, the beads or microtitre plate wells are washed to remove anyunbound components and complex formation is measured either directly orindirectly, for example, as described above. Alternatively, thecomplexes can be dissociated from the matrix, and the level of humanphospholipid scramblase-like binding or activity determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either humanphospholipid scramblase-like protein or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin.Biotinylated human phospholipid scramblase-like molecules or targetmolecules can be prepared from biotin-NHS(N-hydroxy-succinimide) usingtechniques well known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96-well plates (Pierce Chemicals). Alternatively,antibodies reactive with a human phospholipid scramblase-like protein ortarget molecules but which do not interfere with binding of the humanphospholipid scramblase-like protein to its target molecule can bederivatized to the wells of the plate, and unbound target or humanphospholipid scramblase-like protein trapped in the wells by antibodyconjugation. Methods for detecting such complexes, in addition to thosedescribed above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the humanphospholipid scramblase-like protein or target molecule, as well asenzyme-linked assays that rely on detecting an enzymatic activityassociated with the human phospholipid scramblase-like protein or targetmolecule.

In another embodiment, modulators of human phospholipid scramblase-likeexpression are identified in a method in which a cell is contacted witha candidate compound and the expression of human phospholipidscramblase-like mRNA or protein in the cell is determined relative toexpression of human phospholipid scramblase-like mRNA or protein in acell in the absence of the candidate compound. When expression isgreater (statistically significantly greater) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of human phospholipid scramblase-like mRNA orprotein expression. Alternatively, when expression is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of human phospholipid scramblase-like mRNA or proteinexpression. The level of human phospholipid scramblase-like mRNA orprotein expression in the cells can be determined by methods describedherein for detecting human phospholipid scramblase-like mRNA or protein.

In yet another aspect of the invention, the human phospholipidscramblase-like proteins can be used as “bait proteins” in a two-hybridassay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervoset al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.268:12046-12054; Bartel et al. (1993) Bio/Techniques 14:920-924;Iwabuchi et al. (1993) Oncogene 8:1693-1696; and PCT Publication No. WO94/10300), to identify other proteins, which bind to or interact withhuman phospholipid scramblase-like protein (“human phospholipidscramblase-like-binding proteins” or “human phospholipidscramblase-like-bp”) and modulate human phospholipid scramblase-likeactivity. Such human phospholipid scramblase-like-binding proteins arealso likely to be involved in the propagation of signals by the humanphospholipid scramblase-like proteins as, for example, upstream ordownstream elements of the human phospholipid scramblase-like pathway.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(1) map their respective genes on a chromosome; (2) identify anindividual from a minute biological sample (tissue typing); and (3) aidin forensic identification of a biological sample. These applicationsare described in the subsections below.

1. Chromosome Mapping

The isolated complete or partial human phospholipid scramblase-like genesequences of the invention can be used to map their respective humanphospholipid scramblase-like genes on a chromosome, thereby facilitatingthe location of gene regions associated with genetic disease. Computeranalysis of human phospholipid scramblase-like sequences can be used torapidly select PCR primers (preferably 15-25 bp in length) that do notspan more than one exon in the genomic DNA, thereby simplifying theamplification process. These primers can then be used for PCR screeningof somatic cell hybrids containing individual human chromosomes. Onlythose hybrids containing the human gene corresponding to the humanphospholipid scramblase-like sequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow (because they lack a particular enzyme), but in whichhuman cells can, the one human chromosome that contains the geneencoding the needed enzyme will be retained. By using various media,panels of hybrid cell lines can be established. Each cell line in apanel contains either a single human chromosome or a small number ofhuman chromosomes, and a full set of mouse chromosomes, allowing easymapping of individual genes to specific human chromosomes (D'Eustachioet al. (1983) Science 220:919-924). Somatic cell hybrids containing onlyfragments of human chromosomes can also be produced by using humanchromosomes with translocations and deletions.

Other mapping strategies that can similarly be used to map a humanphospholipid scramblase-like sequence to its chromosome include in situhybridization (described in Fan et al. (1990) Proc. Natl. Acad. Sci. USA87:6223-27), pre-screening with labeled flow-sorted chromosomes, andpre-selection by hybridization to chromosome specific cDNA libraries.Furthermore, fluorescence in situ hybridization (FISH) of a DNA sequenceto a metaphase chromosomal spread can be used to provide a precisechromosomal location in one step. For a review of this technique, seeVerma et al. (1988) Human Chromosomes: A Manual of Basic Techniques(Pergamon Press, NY). The FISH technique can be used with a DNA sequenceas short as 500 or 600 bases. However, clones larger than 1,000 baseshave a higher likelihood of binding to a unique chromosomal locationwith sufficient signal intensity for simple detection. Preferably 1,000bases, and more preferably 2,000 bases will suffice to get good resultsin a reasonable amount of time.

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Another strategy to map the chromosomal location of human phospholipidscramblase-like genes uses human phospholipid scramblase-likepolypeptides and fragments and sequences of the present invention andantibodies specific thereto. This mapping can be carried out byspecifically detecting the presence of a human phospholipidscramblase-like polypeptide in members of a panel of somatic cellhybrids between cells of a first species of animal from which theprotein originates and cells from a second species of animal, and thendetermining which somatic cell hybrid(s) expresses the polypeptide andnoting the chromosomes(s) from the first species of animal that itcontains. For examples of this technique, see Pajunen et al. (1988)Cytogenet. Cell. Genet. 47:37-41 and Van Keuren et al. (1986) Hum.Genet. 74:34-40. Alternatively, the presence of a human phospholipidscramblase-like polypeptide in the somatic cell hybrids can bedetermined by assaying an activity or property of the polypeptide, forexample, enzymatic activity, as described in Bordelon-Riser et al.(1979) Somatic Cell Genetics 5:597-613 and Owerbach et al. (1978) Proc.Natl. Acad. Sci. USA 75:5640-5644.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship betweengenes and disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, e.g., Egeland et al. (1987) Nature325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the human phospholipidscramblase-like gene can be determined. If a mutation is observed insome or all of the affected individuals but not in any unaffectedindividuals, then the mutation is likely to be the causative agent ofthe particular disease. Comparison of affected and unaffectedindividuals generally involves first looking for structural alterationsin the chromosomes such as deletions' or translocations that are visiblefrom chromosome spreads or detectable using PCR based on that DNAsequence. Ultimately, complete sequencing of genes from severalindividuals can be performed to confirm the presence of a mutation andto distinguish mutations from polymorphisms.

2. Tissue Typing

The human phospholipid scramblase-like sequences of the presentinvention can also be used to identify individuals from minutebiological samples. The United States military, for example, isconsidering the use of restriction fragment length polymorphism (RFLP)for identification of its personnel. In this technique, an individual'sgenomic DNA is digested with one or more restriction enzymes and probedon a Southern blot to yield unique bands for identification. Thesequences of the present invention are useful as additional DNA markersfor RFLP (described in U.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique for determining the actual base-by-baseDNA sequence of selected portions of an individual's genome. Thus, thehuman phospholipid scramblase-like sequences of the invention can beused to prepare two PCR primers from the 5′ and 3′ ends of thesequences. These primers can then be used to amplify an individual's DNAand subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The human phospholipid scramblase-like sequences of theinvention uniquely represent portions of the human genome. Allelicvariation occurs to some degree in the coding regions of thesesequences, and to a greater degree in the noncoding regions. It isestimated that allelic variation between individual humans occurs with afrequency of about once per each 500 bases. Each of the sequencesdescribed herein can, to some degree, be used as a standard againstwhich DNA from an individual can be compared for identificationpurposes. The noncoding sequences of SEQ ID NO:16 can comfortablyprovide positive individual identification with a panel of perhaps 10 to1,000 primers that each yield a noncoding amplified sequence of 100bases. If a predicted coding sequence, such as that in SEQ ID NO:16 orSEQ ID NO:18, is used, a more appropriate number of primers for positiveindividual identification would be 500 to 2,000.

3. Use of Partial Human Phospholipid Scramblase-Like Sequences inForensic Biology

DNA-based identification techniques can also be used in forensicbiology. In this manner, PCR technology can be used to amplify DNAsequences taken from very small biological samples such as tissues,e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen foundat a crime scene. The amplified sequence can then be compared to astandard, thereby allowing identification of the origin of thebiological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” that is unique to a particular individual. Asmentioned above, actual base sequence information can be used foridentification as an accurate alternative to patterns formed byrestriction enzyme generated fragments. Sequences targeted to noncodingregions of SEQ ID NO:16 are particularly appropriate for this use asgreater numbers of polymorphisms occur in the noncoding regions, makingit easier to differentiate individuals using this technique. Examples ofpolynucleotide reagents include the human phospholipid scramblase-likesequences or portions thereof, e.g., fragments derived from thenoncoding regions of SEQ ID NO:16 having a length of at least 20 or 30bases.

The human phospholipid scramblase-like sequences described herein canfurther be used to provide polynucleotide reagents, e.g., labeled orlabelable probes that can be used in, for example, an in situhybridization technique, to identify a specific tissue. This can be veryuseful in cases where a forensic pathologist is presented with a tissueof unknown origin. Panels of such human phospholipid scramblase-likeprobes, can be used to identify tissue by species and/or by organ type.

In a similar fashion, these reagents, e.g., human phospholipidscramblase-like primers or probes can be used to screen tissue culturefor contamination (i.e., screen for the presence of a mixture ofdifferent types of cells in a culture).

C. Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trails are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. These applications aredescribed in the subsections below.

1. Diagnostic Assays

One aspect of the present invention relates to diagnostic assays fordetecting human phospholipid scramblase-like protein and/or nucleic acidexpression as well as human phospholipid scramblase-like activity, inthe context of a biological sample. An exemplary method for detectingthe presence or absence of human phospholipid scramblase-like proteinsin a biological sample involves obtaining a biological sample from atest subject and contacting the biological sample with a compound or anagent capable of detecting human phospholipid scramblase-like protein ornucleic acid (e.g., mRNA, genomic DNA) that encodes human phospholipidscramblase-like protein such that the presence of human phospholipidscramblase-like protein is detected in the biological sample. Resultsobtained with a biological sample from the test subject may be comparedto results obtained with a biological sample from a control subject.

“Misexpression or aberrant expression”, as used herein, refers to anon-wild type pattern of gene expression, at the RNA or protein level.It includes: expression at non-wild type levels, i.e., over or underexpression; a pattern of expression that differs from wild type in termsof the time or stage at which the gene is expressed, e.g., increased ordecreased expression (as compared with wild type) at a predetermineddevelopmental period or stage; a pattern of expression that differs fromwild type in terms of decreased expression (as compared with wild type)in a predetermined cell type or tissue type; a pattern of expressionthat differs from wild type in terms of the splicing size, amino acidsequence, post-transitional modification, or biological activity of theexpressed polypeptide; a pattern of expression that differs from wildtype in terms of the effect of an environmental stimulus orextracellular stimulus on expression of the gene, e.g., a pattern ofincreased or decreased expression (as compared with wild type) in thepresence of an increase or decrease in the strength of the stimulus.

A preferred agent for detecting human phospholipid scramblase-like mRNAor genomic DNA is a labeled nucleic acid probe capable of hybridizing tohuman phospholipid scramblase-like mRNA or genomic DNA. The nucleic acidprobe can be, for example, a full-length human phospholipidscramblase-like nucleic acid, such as the nucleic acid of SEQ ID NO:16,or a portion thereof, such as a nucleic acid molecule of at least 15,30, 50, 100, 250, or 500 nucleotides in length and sufficient tospecifically hybridize under stringent conditions to human phospholipidscramblase-like mRNA or genomic DNA. Other suitable probes for use inthe diagnostic assays of the invention are described herein.

A preferred agent for detecting human phospholipid scramblase-likeprotein is an antibody capable of binding to human phospholipidscramblase-like protein, preferably an antibody with a detectable label.Antibodies can be polyclonal, or more preferably, monoclonal. An intactantibody, or a fragment thereof (e.g., Fab or F(ab′)₂) can be used. Theterm “labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin.

The term “biological sample” is intended to include tissues, cells, andbiological fluids isolated from a subject, as well as tissues, cells,and fluids present within a subject. That is, the detection method ofthe invention can be used to detect human phospholipid scramblase-likemRNA, protein, or genomic DNA in a biological sample in vitro as well asin vivo. For example, in vitro techniques for detection of humanphospholipid scramblase-like mRNA include Northern hybridizations and insitu hybridizations. In vitro techniques for detection of humanphospholipid scramblase-like protein include enzyme linked immunosorbentassays (ELISAs), Western blots, immunoprecipitations, andimmunofluorescence. In vitro techniques for detection of humanphospholipid scramblase-like genomic DNA include Southernhybridizations. Furthermore, in vivo techniques for detection of humanphospholipid scramblase-like protein include introducing into a subjecta labeled anti-human phospholipid scramblase-like antibody. For example,the antibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a peripheral blood leukocytesample isolated by conventional means from a subject.

The invention also encompasses kits for detecting the presence of humanphospholipid scramblase-like proteins in a biological sample (a testsample). Such kits can be used to determine if a subject is sufferingfrom or is at increased risk of developing a disorder associated withaberrant expression of human phospholipid scramblase-like protein suchas for Scott syndrome, a disorder in platelet clotting, or liverfibrosis. For example, the kit can comprise a labeled compound or agentcapable of detecting human phospholipid scramblase-like protein or mRNAin a biological sample and means for determining the amount of a humanphospholipid scramblase-like protein in the sample (e.g., an anti-humanphospholipid scramblase-like antibody or an oligonucleotide probe thatbinds to DNA encoding a human phospholipid scramblase-like protein,e.g., SEQ ID NO:17). Kits can also include instructions for observingthat the tested subject is suffering from or is at risk of developing adisorder associated with aberrant expression of human phospholipidscramblase-like sequences if the amount of human phospholipidscramblase-like protein or mRNA is above or below a normal level.

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) that binds to humanphospholipid scramblase-like protein; and, optionally, (2) a second,different antibody that binds to human phospholipid scramblase-likeprotein or the first antibody and is conjugated to a detectable agent.For oligonucleotide-based kits, the kit can comprise, for example: (1)an oligonucleotide, e.g., a detectably labeled oligonucleotide, thathybridizes to a human phospholipid scramblase-like nucleic acid sequenceor (2) a pair of primers useful for amplifying a human phospholipidscramblase-like nucleic acid molecule.

The kit can also comprise, e.g., a buffering agent, a preservative, or aprotein stabilizing agent. The kit can also comprise componentsnecessary for detecting the detectable agent (e.g., an enzyme or asubstrate). The kit can also contain a control sample or a series ofcontrol samples that can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container, and all of the various containers are within asingle package along with instructions for observing whether the testedsubject is suffering from or is at risk of developing a disorderassociated with aberrant expression of human phospholipidscramblase-like proteins.

2. Other Diagnostic Assays

In another aspect, the invention features a method of analyzing aplurality of capture probes. The method can be used, e.g., to analyzegene expression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g., anucleic acid or peptide sequence; contacting the array with aphospholipid scramblase-like nucleic acid, preferably purified,polypeptide, preferably purified, or antibody, and thereby evaluatingthe plurality of capture probes. Binding, e.g., in the case of a nucleicacid, hybridization, with a capture probe at an address of theplurality, is detected, e.g., by signal generated from a label attachedto the phospholipid scramblase-like nucleic acid, polypeptide, orantibody. The capture probes can be a set of nucleic acids from aselected sample, e.g., a sample of nucleic acids derived from a controlor non-stimulated tissue or cell.

The method can include contacting the phospholipid scramblase-likenucleic acid, polypeptide, or antibody with a first array having aplurality of capture probes and a second array having a differentplurality of capture probes. The results of each hybridization can becompared, e.g., to analyze differences in expression between afirst andsecond sample. The first plurality of capture probes can be from acontrol sample, e.g., a wild type, normal, or non-diseased,non-stimulated, sample, e.g., a biological fluid, tissue, or cellsample. The second plurality of capture probes can be from anexperimental sample, e.g., a mutant type, at risk, disease-state ordisorder-state, or stimulated, sample, e.g., a biological fluid, tissue,or cell sample.

The plurality of capture probes can be a plurality of nucleic acidprobes each of which specifically hybridizes, with an allele of aphospholipid scramblase-like sequence of the invention. Such methods canbe used to diagnose a subject, e.g., to evaluate risk for a disease ordisorder, to evaluate suitability of a selected treatment for a subject,to evaluate whether a subject has a disease or disorder. Thus, forexample, the 32621 sequence set forth in SEQ ID NO:16 encodes aphospholipid scramblase-like polypeptide that is associated with bloodcoagulation, thus it is useful for evaluating bleeding disorders.

The method can be used to detect single nucleotide polymorphisms (SNPs),as described below.

In another aspect, the invention features a method of analyzing aplurality of probes. The method is useful, e.g., for analyzing geneexpression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the pluralityhaving a unique capture probe, e.g., wherein the capture probes are froma cell or subject which express a phospholipid scramblase-likepolypeptide of the invention or from a cell or subject in which aphospholipid scramblase-like mediated response has been elicited, e.g.,by contact of the cell with a phospholipid scramblase-like nucleic acidor protein of the invention, or administration to the cell or subject aphospholipid scramblase-like nucleic acid or protein of the invention;contacting the array with one or more inquiry probes, wherein an inquiryprobe can be a nucleic acid, polypeptide, or antibody (which ispreferably other than a phospholipid scramblase-like nucleic acid,polypeptide, or antibody of the invention); providing a two dimensionalarray having a plurality of addresses, each address of the pluralitybeing positionally distinguishable from each other address of theplurality, and each address of the plurality having a unique captureprobe, e.g., wherein the capture probes are from a cell or subject whichdoes not express a phospholipid scramblase-like sequence of theinvention (or does not express as highly as in the case of thephospholipid scramblase-like positive plurality of capture probes) orfrom a cell or subject in which a phospholipid scramblase-like-mediatedresponse has not been elicited (or has been elicited to a lesser extentthan in the first sample); contacting the array with one or more inquiryprobes (which is preferably other than a phospholipid scramblase-likenucleic acid, polypeptide, or antibody of the invention), and therebyevaluating the plurality of capture probes. Binding, e.g., in the caseof a nucleic acid, hybridization, with a capture probe at an address ofthe plurality, is detected, e.g., by signal generated from a labelattached to the nucleic acid, polypeptide, or antibody.

In another aspect, the invention features a method of analyzing aphospholipid scramblase-like sequence of the invention, e.g., analyzingstructure, function, or relatedness to other nucleic acid or amino acidsequences. The method includes: providing a phospholipid scramblase-likenucleic acid or amino acid sequence, e.g., the 32621 sequence set forthin SEQ ID NO:17 or a portion thereof; comparing the phospholipidscramblase-like sequence with one or more preferably a plurality ofsequences from a collection of sequences, e.g., a nucleic acid orprotein sequence database; to thereby analyze the phospholipidscramblase-like sequence of the invention.

The method can include evaluating the sequence identity between aphospholipid scramblase-like sequence of the invention, e.g., the 32621sequence, and a database sequence. The method can be performed byaccessing the database at a second site, e.g., over the internet.

In another aspect, the invention features, a set of oligonucleotides,useful, e.g., for identifying SNP's, or identifying specific alleles ofa phospholipid scramblase-like sequence of the invention, e.g., the32621 sequence. The set includes a plurality of oligonucleotides, eachof which has a different nucleotide at an interrogation position, e.g.,an SNP or the site of a mutation. In a preferred embodiment, theoligonucleotides of the plurality identical in sequence with one another(except for differences in length). The oligonucleotides can be providedwith differential labels, such that an oligonucleotides which hybridizesto one allele provides a signal that is distinguishable from anoligonucleotides which hybridizes to a second allele.

3. Prognostic Assays

The methods described herein can furthermore be utilized as diagnosticor prognostic assays to identify subjects having or at risk ofdeveloping a disease or disorder associated with human phospholipidscramblase-like protein, human phospholipid scramblase-like nucleic acidexpression, or human phospholipid scramblase-like activity. Prognosticassays can be used for prognostic or predictive purposes to therebyprophylactically treat an individual prior to the onset of a disordercharacterized by or associated with human phospholipid scramblase-likeprotein, human phospholipid scramblase-like nucleic acid expression, orhuman phospholipid scramblase-like activity.

Thus, the present invention provides a method in which a test sample isobtained from a subject, and human phospholipid scramblase-like proteinor nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein thepresence of human phospholipid scramblase-like protein or nucleic acidis diagnostic for a subject having or at risk of developing a disease ordisorder associated with aberrant human phospholipid scramblase-likeexpression or activity. As used herein, a “test sample” refers to abiological sample obtained from a subject of interest. For example, atest sample can be a biological fluid (e.g., serum), cell sample, ortissue.

Furthermore, using the prognostic assays described herein, the presentinvention provides methods for determining whether a subject can beadministered a specific agent (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, nucleic acid, small molecule, or otherdrug candidate) or class of agents (e.g., agents of a type that decreasehuman phospholipid scramblase-like activity) to effectively treat adisease or disorder associated with aberrant human phospholipidscramblase-like expression or activity. In this manner, a test sample isobtained and human phospholipid scramblase-like protein or nucleic acidis detected. The presence of human phospholipid scramblase-like proteinor nucleic acid is diagnostic for a subject that can be administered theagent to treat a disorder associated with aberrant human phospholipidscramblase-like expression or activity.

The methods of the invention can also be used to detect genetic lesionsor mutations in a human phospholipid scramblase-like gene, therebydetermining if a subject with the lesioned gene is at risk for adisorder characterized by abnormal platelet disfunction or clotting orsome other immune or hemopoetic disorder. In preferred embodiments, themethods include detecting, in a sample of cells from the subject, thepresence or absence of a genetic lesion or mutation characterized by atleast one of an alteration affecting the integrity of a gene encoding ahuman phospholipid scramblase-like-protein, or the misexpression of thehuman phospholipid scramblase-like gene. For example, such geneticlesions or mutations can be detected by ascertaining the existence of atleast one of: (1) a deletion of one or more nucleotides from a humanphospholipid scramblase-like gene; (2) an addition of one or morenucleotides to a human phospholipid scramblase-like gene; (3) asubstitution of one or more nucleotides of a human phospholipidscramblase-like gene; (4) a chromosomal rearrangement of a humanphospholipid scramblase-like gene; (5) an alteration in the level of amessenger RNA transcript of a human phospholipid scramblase-like gene;(6) an aberrant modification of a human phospholipid scramblase-likegene, such as of the methylation pattern of the genomic DNA; (7) thepresence of a non-wild-type splicing pattern of a messenger RNAtranscript of a human phospholipid scramblase-like gene; (8) anon-wild-type level of a human phospholipid scramblase-like-protein; (9)an allelic loss of a human phospholipid scramblase-like gene; and (10)an inappropriate post-translational modification of a human phospholipidscramblase-like-protein. As described herein, there are a large numberof assay techniques known in the art that can be used for detectinglesions in a human phospholipid scramblase-like gene. Any cell type ortissue, preferably peripheral blood cells, in which human phospholipidscramblase-like proteins are expressed may be utilized in the prognosticassays described herein.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the humanphospholipid scramblase-like-gene (see, e.g., Abravaya et al. (1995)Nucleic Acids Res. 23:675-682). It is anticipated that PCR and/or LCRmay be desirable to use as a preliminary amplification step inconjunction with any of the techniques used for detecting mutationsdescribed herein.

Alternative amplification methods include self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a human phospholipidscramblase-like gene from a sample cell can be identified by alterationsin restriction enzyme cleavage patterns of isolated test sample andcontrol DNA digested with one or more restriction endonucleases.Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Pat.No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in a human phospholipidscramblase-like molecule can be identified by hybridizing a sample andcontrol nucleic acids, e.g., DNA or RNA, to high density arrayscontaining hundreds or thousands of oligonucleotides probes (Cronin etal. (1996) Human Mutation 7:244-255; Kozal et al. (1996) Nature Medicine2:753-759). In yet another embodiment, any of a variety of sequencingreactions known in the art can be used to directly sequence the humanphospholipid scramblase-like gene and detect mutations by comparing thesequence of the sample human phospholipid scramblase-like gene with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxim andGilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977)Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any ofa variety of automated sequencing procedures can be utilized whenperforming the diagnostic assays ((1995) Bio/Techniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT PublicationNo. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; andGriffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

Other methods for detecting mutations in the human phospholipidscramblase-like gene include methods in which protection from cleavageagents is used to detect mismatched bases in RNA/RNA or RNA/DNAheteroduplexes (Myers et al. (1985) Science 230:1242). See also Cottonet al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992)Methods Enzymol. 217:286-295. In a preferred embodiment, the control DNAor RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more “DNA mismatch repair” enzymes that recognize mismatched basepairs in double-stranded DNA in defined systems for detecting andmapping point mutations in human phospholipid scramblase-like cDNAsobtained from samples of cells. See, e.g., Hsu et al. (1994)Carcinogenesis 15:1657-1662. According to an exemplary embodiment, aprobe based on a human phospholipid scramblase-like sequence, e.g., awild-type human phospholipid scramblase-like sequence, is hybridized toa cDNA or other DNA product from a test cell(s). The duplex is treatedwith a DNA mismatch repair enzyme, and the cleavage products, if any,can be detected from electrophoresis protocols or the like. See, e.g.,U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in human phospholipid scramblase-like genes.For example, single-strand conformation polymorphism (SSCP) may be usedto detect differences in electrophoretic mobility between mutant andwild-type nucleic acids (Orita et al. (1989) Proc. Natl. Acad. Sci. USA86:2766; see also Cotton (1993) Mutat. Res. 285:125-144; Hayashi (1992)Genet. Anal. Tech. Appl. 9:73-79). The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double-stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditions thatpermit hybridization only if a perfect match is found (Saiki et al.(1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci. USA86:6230). Such allele-specific oligonucleotides are hybridized toPCR-amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele-specific amplification technology, which dependson selective PCR amplification, may be used in conjunction with theinstant invention. Oligonucleotides used as primers for specificamplification may carry the mutation of interest in the center of themolecule so that amplification depends on differential hybridization(Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme3′ end of one primer where, under appropriate conditions, mismatch canprevent or reduce polymerase extension (Prossner (1993) Tibtech 11:238).In addition, it may be desirable to introduce a novel restriction sitein the region of the mutation to create cleavage-based detection(Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated thatin certain embodiments amplification may also be performed using Taqligase for amplification (Barany (1991) Proc. Natl. Acad. Sci. USA88:189). In such cases, ligation will occur only if there is a perfectmatch at the 3′ end of the 5′ sequence making it possible to detect thepresence of a known mutation at a specific site by looking for thepresence or absence of amplification.

The methods described herein may be performed, for example, by utilizingprepackaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnosed patients exhibiting symptoms orfamily history of a disease or illness involving a human phospholipidscramblase-like gene.

4. Pharmacogenomics

Agents, or modulators that have a stimulatory or inhibitory effect onhuman phospholipid scramblase-like activity (e.g., human phospholipidscramblase-like gene expression) as identified by a screening assaydescribed herein, can be administered to individuals to treat(prophylactically or therapeutically) disorders associated with aberranthuman phospholipid scramblase-like activity as well as to modulate thephenotype of an immune, hemopeotic, or blood clotting response. Inconjunction with such treatment, the pharmacogenomics (i.e., the studyof the relationship between an individual's genotype and thatindividual's response to a foreign compound or drug) of the individualmay be considered. Differences in metabolism of therapeutics can lead tosevere toxicity or therapeutic failure by altering the relation betweendose and blood concentration of the pharmacologically active drug. Thus,the pharmacogenomics of the individual permits the selection ofeffective agents (e.g., drugs) for prophylactic or therapeutictreatments based on a consideration of the individual's genotype. Suchpharmacogenomics can further be used to determine appropriate dosagesand therapeutic regimens. Accordingly, the activity of humanphospholipid scramblase-like protein, expression of human phospholipidscramblase-like nucleic acid, or mutation content of human phospholipidscramblase-like genes in an individual can be determined to therebyselect appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Linder (1997) Clin. Chem.43(2):254-266. In general, two types of pharmacogenetic conditions canbe differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body are referred to as “altered drugaction.” Genetic conditions transmitted as single factors altering theway the body acts on drugs are referred to as “altered drug metabolism”.These pharmacogenetic conditions can occur either as rare defects or aspolymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency(G6PD) is a common inherited enzymopathy in which the main clinicalcomplication is haemolysis after ingestion of oxidant drugs(antimalarials, sulfonamides, analgesics, nitrofurans) and consumptionof fava beans.

Differences in metabolism of therapeutics can lead to severe toxicity ortherapeutic failure by altering the relation between dose and bloodconcentration of the pharmacologically active drug. Thus, a physician orclinician may consider applying knowledge obtained in relevantpharmacogenomics studies in determining whether to administer aphospholipid scramblase-like molecule or phospholipid scramblase-likemodulator of the invention as well as tailoring the dosage and/ortherapeutic regimen of treatment with a phospholipid scramblase-likemolecule or phospholipid scramblase-like modulator of the invention.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, an “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drug's target is known (e.g., aphospholipid scramblase-like protein of the present invention), allcommon variants of that gene can be fairly easily identified in thepopulation and it can be determined if having one version of the geneversus another is associated with a particular drug response.

Alternatively, a method termed the “gene expression profiling”, can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., a phospholipidscramblase-like molecule or phospholipid scramblase-like modulator ofthe present invention) can give an indication whether gene pathwaysrelated to toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment of an individual.This knowledge, when applied to dosing or drug selection, can avoidadverse reactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a phospholipidscramblase-like molecule or phospholipid scramblase-like modulator ofthe invention, such as a modulator identified by one of the exemplaryscreening assays described herein.

The present invention further provides methods for identifying newagents, or combinations, that are based on identifying agents thatmodulate the activity of one or more of the gene products encoded by oneor more of the phospholipid scramblase-like genes of the presentinvention, wherein these products may be associated with resistance ofthe cells to a therapeutic agent. Specifically, the activity of theproteins encoded by the phospholipid scramblase-like genes of thepresent invention can be used as a basis for identifying agents forovercoming agent resistance. By blocking the activity of one or more ofthe resistance proteins, target cells, e.g., hepatic stellate cells,will become sensitive to treatment with an agent that the unmodifiedtarget cells were resistant to.

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a phospholipid scramblase-like protein can be applied inclinical trials. For example, the effectiveness of an agent determinedby a screening assay as described herein to increase phospholipidscramblase-like gene expression, protein levels, or upregulatePhospholipid scramblase-like activity, can be monitored in clinicaltrials of subjects exhibiting decreased phospholipid scramblase-likegene expression, protein levels, or downregulated phospholipidscramblase-like activity. Alternatively, the effectiveness of an agentdetermined by a screening assay to decrease phospholipid scramblase-likegene expression, protein levels, or downregulate phospholipidscramblase-like activity, can be monitored in clinical trials ofsubjects exhibiting increased phospholipid scramblase-like geneexpression, protein levels, or upregulated phospholipid scramblase-likeactivity. In such clinical trials, the expression or activity of aphospholipid scramblase-like gene, and preferably, other genes that havebeen implicated in, for example, a phospholipidscramblase-like-associated disorder can be used as a “read out” ormarkers of the phenotype of a particular cell.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, a PM will show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the activity of human phospholipid scramblase-like protein,expression of human phospholipid scramblase-like nucleic acid, ormutation content of human phospholipid scramblase-like genes in anindividual can be determined to thereby select appropriate agent(s) fortherapeutic or prophylactic treatment of the individual. In addition,pharmacogenetic studies can be used to apply genotyping of polymorphicalleles encoding drug-metabolizing enzymes to the identification of anindividual's drug responsiveness phenotype. This knowledge, when appliedto dosing or drug selection, can avoid adverse reactions or therapeuticfailure and thus enhance therapeutic or prophylactic efficiency whentreating a subject with a human phospholipid scramblase-like modulator,such as a modulator identified by one of the exemplary screening assaysdescribed herein.

5. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of human phospholipid scramblase-like genes canbe applied not only in basic drug screening but also in clinical trials.For example, the effectiveness of an agent, as determined by a screeningassay as described herein, to increase or decrease human phospholipidscramblase-like gene expression, protein levels, or protein activity,can be monitored in clinical trials of subjects exhibiting decreased orincreased human phospholipid scramblase-like gene expression, proteinlevels, or protein activity. In such clinical trials, human phospholipidscramblase-like expression or activity and preferably that of othergenes that have been implicated in for example, a cellular proliferationdisorder, can be used as a marker of the immune responsiveness of aparticular cell.

For example, and not by way of limitation, genes that are modulated incells by treatment with an agent (e.g., compound, drug, or smallmolecule) that modulates human phospholipid scramblase-like activity(e.g., as identified in a screening assay described herein) can beidentified. Thus, to study the effect of agents disorders, for example,in a clinical trial, cells can be isolated and RNA prepared and analyzedfor the levels of expression of human phospholipid scramblase-like genesand other genes implicated in the disorder. The levels of geneexpression (i.e., a gene expression pattern) can be quantified byNorthern blot analysis or RT-PCR, as described herein, or alternativelyby measuring the amount of protein produced, by one of the methods asdescribed herein, or by measuring the levels of activity of humanphospholipid scramblase-like genes or other genes. In this way, the geneexpression pattern can serve as a marker, indicative of thephysiological response of the cells to the agent. Accordingly, thisresponse state may be determined before, and at various points during,treatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (1) obtaininga preadministration sample from a subject prior to administration of theagent; (2) detecting the level of expression of a human phospholipidscramblase-like protein, mRNA, or genomic DNA in the preadministrationsample; (3) obtaining one or more postadministration samples from thesubject; (4) detecting the level of expression or activity of the humanphospholipid scramblase-like protein, mRNA, or genomic DNA in thepostadministration samples; (5) comparing the level of expression oractivity of the human phospholipid scramblase-like protein, mRNA, orgenomic DNA in the preadministration sample with the human phospholipidscramblase-like protein, mRNA, or genomic DNA in the postadministrationsample or samples; and (vi) altering the administration of the agent tothe subject accordingly to bring about the desired effect, i.e., forexample, an increase or a decrease in the expression or activity of ahuman phospholipid scramblase-like protein.

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant human phospholipidscramblase-like expression or activity. “Subject”, as used herein, canrefer to a mammal, e.g., a human, or to an experimental or animal ordisease model. The subject can also be a non-human animal, e.g., ahorse, cow, goat, or other domestic animal. Additionally, thecompositions of the invention find use in the treatment of disordersdescribed herein. Thus, therapies for disorders associated with aberranthuman phospholipid scramblase activity are encompassed herein.“Treatment” is herein defined as the application or administration of atherapeutic agent to a patient, or application or administration of atherapeutic agent to an isolated tissue or cell line from a patient, whohas a disease, a symptom of disease or a predisposition toward adisease, with the purpose to cure, heal, alleviate, relieve, alter,remedy, ameliorate, improve or affect the disease, the symptoms ofdisease or the predisposition toward disease. A “therapeutic agent”includes, but is not limited to, small molecules, peptides, antibodies,ribozymes and antisense oligonucleotides.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject a disease or condition associated with an aberrant humanphospholipid scramblase-like expression or activity by administering tothe subject an agent that modulates human phospholipid scramblase-likeexpression or at least one human phospholipid scramblase-like geneactivity. Subjects at risk for a disease that is caused, or contributedto, by aberrant human phospholipid scramblase-like expression oractivity can be identified by, for example, any or a combination ofdiagnostic or prognostic assays as described herein. Administration of aprophylactic agent can occur prior to the manifestation of symptomscharacteristic of the human phospholipid scramblase-like aberrancy, suchthat a disease or disorder is prevented or, alternatively, delayed inits progression. Depending on the type of human phospholipidscramblase-like aberrancy, for example, a human phospholipidscramblase-like agonist or human phospholipid scramblase-like antagonistagent can be used for treating the subject. The appropriate agent can bedetermined based on screening assays described herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating humanphospholipid scramblase-like expression or activity for therapeuticpurposes. The modulatory method of the invention involves contacting acell with an agent that modulates one or more of the activities of humanphospholipid scramblase-like protein activity associated with the cell.An agent that modulates human phospholipid scramblase-like proteinactivity can be an agent as described herein, such as a nucleic acid ora protein, a naturally-occurring cognate ligand of a human phospholipidscramblase-like protein, a peptide, a human phospholipid scramblase-likepeptidomimetic, or other small molecule. In one embodiment, the agentstimulates one or more of the biological activities of humanphospholipid scramblase-like protein. Examples of such stimulatoryagents include active human phospholipid scramblase-like protein and anucleic acid molecule encoding a human phospholipid scramblase-likeprotein that has been introduced into the cell. In another embodiment,the agent inhibits one or more of the biological activities of humanphospholipid scramblase-like protein. Examples of such inhibitory agentsinclude antisense human phospholipid scramblase-like nucleic acidmolecules and anti-human phospholipid scramblase-like antibodies.

These modulatory methods can be performed in vitro (e.g., by culturingthe cell with the agent) or, alternatively, in vivo (e.g., byadministering the agent to a subject). As such, the present inventionprovides methods of treating an individual afflicted with a disease ordisorder characterized by aberrant expression or activity of a humanphospholipid scramblase-like protein or nucleic acid molecule. In oneembodiment, the method involves administering an agent (e.g., an agentidentified by a screening assay described herein), or a combination ofagents, that modulates (e.g., upregulates or downregulates) humanphospholipid scramblase-like expression or activity. In anotherembodiment, the method involves administering a human phospholipidscramblase-like protein or nucleic acid molecule as therapy tocompensate for reduced or aberrant human phospholipid scramblase-likeexpression or activity.

Stimulation of human phospholipid scramblase-like activity is desirablein situations in which a human phospholipid scramblase-like protein isabnormally downregulated and/or in which increased human phospholipidscramblase-like activity is likely to have a beneficial effect.Conversely, inhibition of human phospholipid scramblase-like activity isdesirable in situations in which human phospholipid scramblase-likeactivity is abnormally upregulated and/or in which decreased humanphospholipid scramblase-like activity is likely to have a beneficialeffect.

This invention is further illustrated by the following examples, whichshould not be construed as limiting.

EXPERIMENTAL Example 1 Identification and Characterization of 32621Human Scramblase

The human 32621-like sequence (SEQ ID NO:16), which is approximately1542 nucleotides long including untranslated regions, contains apredicted methionine-initiated coding sequence of about 990 nucleotides(nucleotides 156-1145 of SEQ ID NO:16; SEQ ID NO:18). The codingsequence encodes a 329 amino acid protein (SEQ ID NO:17).

A search of the nucleotide and protein databases revealed that 32621encodes a precursor polypeptide that shares similarity with severalphospholipid scramblase proteins. An alignment of the protein sequenceshaving highest similarity to the 32621 precursor polypeptide is shown inFIG. 21. The alignment was generated using the Clustal method withPAM250 residue weight table and sequence identities were determined byFASTA (Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA85:2444-2448).

The 32621 protein displays similarity (approximately 45% identity overthe full amino acid sequence) to the murine phospholipid scramblase-likepolypeptide (SEQ ID NO:19; SP Accession No. 2935163; Zhou et al (1998)Biochem. 37:2356-2360 (see FIG. 21). It also displays similarity to thehuman Mm-1 cell derived transplantability-associated gene 1b(approximately 41% identity over the full amino acid sequence; SEQ IDNO:20; SP Accession No. 3510297; Kasukabe et al. (1998) Biochem.Biophys. Res. Comm. 249:449-455 (see FIG. 21).

Example 2 Tissue Distribution of 32621 mRNA

Expression levels of 32621 in various tissue and cell types weredetermined by quantitative RT-PCR (Reverse Transcriptase PolymeraseChain Reaction; Taqman® brand PCR kit, Applied Biosystems). Thequantitative RT-PCR reactions were performed according to the kitmanufacturer's instructions. The results of the Taqman® analysis areshown in FIGS. 24A-27.

Northern blot hybridizations with various RNA samples are performedunder standard conditions and washed under stringent conditions, i.e.,0.2×SSC at 65° C. A DNA probe corresponding to all or a portion of the32621 cDNA (SEQ ID NO:16) can be used. The DNA is radioactively labeledwith ³²P-dCTP using the Prime-It Kit (Stratagene, La Jolla, Calif.)according to the instructions of the supplier. Filters containing mRNAfrom mouse hematopoietic and endocrine tissues, and cancer cell lines(Clontech, Palo Alto, Calif.) are probed in ExpressHyb hybridizationsolution (Clontech) and washed at high stringency according tomanufacturer's recommendations.

TaqMan analysis of 32621 revealed expression in a number of tissues,including the following: artery; vein; aortic SMC, smooth muscle cells,early); aortic SMC late; static HUVEC, human umbilical vein endothelialcells; shear HUVEC; heart; heart CHF, congestive heart failure hearttissue; kidney; skeletal muscle; adipose; pancreas; primary osteoblasts;osteoclasts; skin; spinal cord; brain cortex; brain hypothalamus; nerve;DRG, dorsal root ganglion; glial cells, astrocytes; glioblastoma;breast; breast tumor; ovary; ovarian tumor; prostate; prostate tumor;prostate epithelial cells; colon; colon tumor; lung; lung tumor; lungCOPD, chronic obstructive pulmonary diseased lung; colon IBD,inflammatory bowel diseased colon; liver; liver fibrosis; dermal cells;spleen; tonsil; lymph node; thymus; skin-decubitis; synovium; bonemarrow mononuclear cells; and activated peripheral blood mononuclearcells. High expression of 32621 occurred in aortic smooth muscle cells,HUVEC, brain cortex, brain hypothalamus, normal ovary, and fibroticliver cells. See FIGS. 24A-B.

Expression of 32621 was further observed in various cell lines andtissues, including the following: conf HMVEC, microvascular endothelialcells; fetal heart; normal atrium; normal ventricle; heart diseasedventricle; normal kidney; kidney HT; skeletal muscle; skeletal muscle;liver; liver with inflammation; fetal adrenal; Wilms Tumor; spinal cord;and diseased cartilage. Relative expression levels of 32621 were alsodetermined in various liver samples from animals fed modified diets. SeeFIGS. 25A-B and 26.

Expression of 32621 was observed in: aortic smooth muscle cells(ASMC)-A1PO; ASMC-A2P3; ASMC-A3P4; ASMC-AL; coronary artery smoothmuscle cells (CASMC)-C1P3; CASMC-C2P3; CASMC-C5P0; CASMC-C1P6;macrophage cells; macrophage cells treated with interferon γ; CD40+macrophage cells; macrophage cells treated with lipopolysaccharide;HUVEC, human umbilical vein endothelial cells; HMVEC, humanmicrovascular endothelial cells; HAEC1, human aortic endothelial cells;HCAEC3, human coronary arterial endothelial cells; HCRE; RPTE, renalproximal tubule epithelial cells; MC; SKM1, myelogenous leukemia cells;and HLF, hepatocellular carcinoma cell line. Of these cell types, 32621expression was high in microvascular endothelial cells, withmicrovascular endothelial cells exhibiting an expression level about1990 times higher than in macrophage cells. See FIG. 27.

Example 3 Recombinant Expression of 32621 in Bacterial Cells

In this example, the 32621-like sequence is expressed as a recombinantglutathione-S-transferase (GST) fusion polypeptide in E. coli and thefusion polypeptide is isolated and characterized. Specifically, the32621-like sequence is fused to GST and this fusion polypeptide isexpressed in E. coli, e.g., strain PEB199. Expression of theGST-32621-like fusion protein in PEB199 is induced with IPTG. Therecombinant fusion polypeptide is purified from crude bacterial lysatesof the induced PEB199 strain by affinity chromatography on glutathionebeads. Using polyacrylamide gel electrophoretic analysis of thepolypeptide purified from the bacterial lysates, the molecular weight ofthe resultant fusion polypeptide is determined.

Example 4 Expression of Recombinant 32621-Like Protein in COS Cells

To express the 32621-like gene in COS cells, the pcDNA/Amp vector byInvitrogen Corporation (San Diego, Calif.) is used. This vector containsan SV40 origin of replication, an ampicillin resistance gene, an E. colireplication origin, a CMV promoter followed by a polylinker region, andan SV40 intron and polyadenylation site. A DNA fragment encoding theentire 32621-like protein and an HA tag (Wilson et al. (1984) Cell37:767) or a FLAG tag fused in-frame to its 3′ end of the fragment iscloned into the polylinker region of the vector, thereby placing theexpression of the recombinant protein under the control of the CMVpromoter.

To construct the plasmid, the 32621-like DNA sequence is amplified byPCR using two primers. The 5′ primer contains the restriction site ofinterest followed by approximately twenty nucleotides of the 32621-likecoding sequence starting from the initiation codon; the 3′ end sequencecontains complementary sequences to the other restriction site ofinterest, a translation stop codon, the HA tag or FLAG tag and the last20 nucleotides of the 32621-like coding sequence. The PCR amplifiedfragment and the pCDNA/Amp vector are digested with the appropriaterestriction enzymes and the vector is dephosphorylated using the CIAPenzyme (New England Biolabs, Beverly, Mass.). Preferably the tworestriction sites chosen are different so that the 32621-like gene isinserted in the correct orientation. The ligation mixture is transformedinto E. coli cells (strains HB101, DH5α, SURE, available from StratageneCloning Systems, La Jolla, Calif., can be used), the transformed cultureis plated on ampicillin media plates, and resistant colonies areselected. Plasmid DNA is isolated from transformants and examined byrestriction analysis for the presence of the correct fragment.

COS cells are subsequently transfected with the 32621-like-pcDNA/Ampplasmid DNA using the calcium phosphate or calcium chlorideco-precipitation methods, DEAE-dextran-mediated transfection,lipofection, or electroporation. Other suitable methods for transfectinghost cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989. The expression of the 32621-like polypeptide is detected byradiolabelling (³⁵S-methionine or ³⁵S-cysteine available from NEN,Boston, Mass., can be used) and immunoprecipitation (Harlow, E. andLane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1988) using an HA specific monoclonalantibody. Briefly, the cells are labeled for 8 hours with ³⁵S-methionine(or ³⁵S-cysteine). The culture media are then collected and the cellsare lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1%SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culturemedia are precipitated with an HA specific monoclonal antibody.Precipitated polypeptides are then analyzed by SDS-PAGE.

Alternatively, DNA containing the 32621-like coding sequence is cloneddirectly into the polylinker of the pCDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of the32621-like polypeptide is detected by radiolabelling andimmunoprecipitation using a 32621-like specific monoclonal antibody.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

Chapter 5 27802, A Novel Adenylate Kinase BACKGROUND OF THE INVENTION

Adenylate kinases play a key role in the regulation of energy balancewithin cells, particularly maintenance of the ratio of ATP with itsdiphosphate (ADP) and monophosphate forms (AMP). ATP serves as theprimary source of energy for biochemical reactions in cells and is alsoa key precursor in DNA and RNA synthesis during cellular growth andreplication. The energy associated with the terminal phosphate bonds ofATP may be transferred to other nucleotides using a nucleosidemonophosphate kinase such as adenylate kinase. In this manner, theterminal energy-rich phosphate bonds of ATP may be transferred to theappropriate nucleotides for use in a variety of biosynthetic andenergy-requiring processes, such as biosynthesis of macromolecules,active ion transport, muscle contraction, thermogenesis, etc. A numberof these energy-requiring biosynthetic reactions hydrolyze ATP into AMPplus pyrophosphate. Reutilization of the resulting AMP requiresconversion back into the triphosphate form following conversion to ADP.Various nucleotide monophosphate kinases carry out the first step ofphosphorylating AMP to its diphosphate form at the expense of ATP. Inthe case of adenylate kinase, this reversible reaction is given asAMP+ATP≡2 ADP.

Adenylate kinases also play a role in regulating the flow of carbonbetween net accumulation of glucose via the gluconeogenesis pathway andits subsequent catabolism via the glycolytic pathway by way of theircontrol over the ratio of AMP to ATP. AMP is a positive allostericeffector of the enzyme 6-phophofructo-1-kinase, which shifts, and anegative allosteric effector for the enzyme fructose-1,6-bisphosphatase.When the first of these enzymes is activated, carbon flow is shifted inthe direction of glycolysis; when the second of these enzymes isactivated, carbon flow shifts in the direction of gluconeogenesis. Thus,increases in the ratio of AMP to ATP shift carbon flow towardglycolysis, while decreases in the ratio of AMP to ATP shift carbon flowtoward glucose formation.

These enzymes have been studied in a number of mammals, including rat,porcine, chicken, bovine, rabbit, and humans. Evidence from biochemicalstudies suggests that human tissues have five adenylate kinase isozymes,AK1-AK5. Thus far the cDNAs of human AK1, AK2, AK4, and AK5 have beencloned. Adenylate kinase isoforms in humans are sequence related andalso related to UMP/CMP kinases from several species. See Rompay et al.(1999) Eur. J. Biochem. 261:509-516, and the references cited therein.

The adenylate kinase isozymes AK1 (or myokinase), which is a cytosolicenzyme present in brain, skeletal muscle, and erythrocytes, and AK2,which is associated with the mitochondrial membrane in liver, spleen,heart, and kidney, both utilize ATP as their nucleoside triphosphatedonor substrate. AK3 (or GTP:AMP phosphotransferase) is located in themitochondrial matrix, primarily in heart and liver cells, and uses MgGTPinstead of MgATP. AK4 and AK5 are both localized in brain tissue.

Several regions of AK family enzymes are well conserved, including thenucleoside triphosphate binding glycine-rich region, the nucleosidemonophosphate binding site, and the lid domain that closes over thesubstrate upon binding (see Schulz (1987) Cold Spring Harbor Symp.Quant. Biol. 52:429-439).

These enzymes assist with maintenance of energy production andutilization within cells, particularly in cells having high rates ofgrowth and metabolic activity such as in heart, skeletal muscle, andliver. In fact, adenylate kinase deficiency has been linked to hemolyticanemia and neurological disorders such as neurofibromatosis (Xu et al.(1992) Genomics 13:537-542. In addition, targeting regulation of ATPsynthesis has been the basis of antiproliferative drugs for treatment ofviral infections and cancer.

Adenylate kinases are also useful for activating nucleoside analoguesused as pharmaceuticals, especially for cancer and viral infection. Mostof these analogues must be phosphorylated to the triphosphate form inorder to be pharmaceutically active. The first phosphorylation step inthe activation of nucleoside analogs is catalyzed by deoxyribonucleosidekinases. Phosphorylation to the di- and triphosphates are then required.

Accordingly, adenylate kinases are a major target for drug action anddevelopment. Thus, it is valuable to the field of pharmaceuticaldevelopment to identify and characterize previously unknown adenylatekinases. The present invention advances the state of the art byproviding a previously unidentified human adenylate kinase.

SUMMARY OF THE INVENTION

Isolated nucleic acid molecules corresponding to adenylate kinasenucleic acid sequences are provided. Additionally, amino acid sequencescorresponding to the polynucleotides are encompassed. In particular, thepresent invention provides for isolated nucleic acid moleculescomprising nucleotide sequences encoding the amino acid sequence shownin SEQ ID NO:22. Further provided are adenylate kinase polypeptideshaving an amino acid sequence encoded by a nucleic acid moleculedescribed herein.

The present invention also provides vectors and host cells forrecombinant expression of the nucleic acid molecules described herein,as well as methods of making such vectors and host cells and for usingthem for production of the polypeptides or peptides of the invention byrecombinant techniques.

The adenylate kinase molecules of the present invention are useful formodulating cellular growth and/or cellular metabolic pathwaysparticularly for regulating one or more proteins involved in growth andmetabolism. Accordingly, in one aspect, this invention provides isolatednucleic acid molecules encoding adenylate kinase proteins orbiologically active portions thereof, as well as nucleic acid fragmentssuitable as primers or hybridization probes for the detection ofadenylate kinase-encoding nucleic acids.

Another aspect of this invention features isolated or recombinantadenylate kinase proteins and polypeptides. Preferred adenylate kinaseproteins and polypeptides possess at least one biological activitypossessed by naturally occurring adenylate kinase proteins.

Variant nucleic acid molecules and polypeptides substantially homologousto the nucleotide and amino acid sequences set forth in the sequencelistings are encompassed by the present invention. Additionally,fragments and substantially homologous fragments of the nucleotide andamino acid sequences are provided.

Antibodies and antibody fragments that selectively bind the adenylatekinase polypeptides and fragments are provided. Such antibodies areuseful in detecting the adenylate kinase polypeptides as well as inregulating the T-cell immune response and cellular activity,particularly growth and proliferation.

In another aspect, the present invention provides a method for detectingthe presence of adenylate kinase activity or expression in a biologicalsample by contacting the biological sample with an agent capable ofdetecting an indicator of adenylate kinase activity such that thepresence of adenylate kinase activity is detected in the biologicalsample.

In yet another aspect, the invention provides a method for modulatingadenylate kinase activity comprising contacting a cell with an agentthat modulates (inhibits or stimulates) adenylate kinase activity orexpression such that adenylate kinase activity or expression in the cellis modulated. In one embodiment, the agent is an antibody thatspecifically binds to adenylate kinase protein. In another embodiment,the agent modulates expression of adenylate kinase protein by modulatingtranscription of an adenylate kinase gene, splicing of an adenylatekinase mRNA, or translation of an adenylate kinase mRNA. In yet anotherembodiment, the agent is a nucleic acid molecule having a nucleotidesequence that is antisense to the coding strand of the adenylate kinasemRNA or the adenylate kinase gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant adenylatekinase protein activity or nucleic acid expression by administering anagent that is an adenylate kinase modulator to the subject. In oneembodiment, the adenylate kinase modulator is an adenylate kinaseprotein. In another embodiment, the adenylate kinase modulator is anadenylate kinase nucleic acid molecule. In other embodiments, theadenylate kinase modulator is a peptide, peptidomimetic, or other smallmolecule.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic lesion or mutation characterized byat least one of the following: (1) aberrant modification or mutation ofa gene encoding an adenylate kinase protein; (2) misregulation of a geneencoding an adenylate kinase protein; and (3) aberrantpost-translational modification of an adenylate kinase protein, whereina wild-type form of the gene encodes a protein with an adenylate kinaseactivity.

In another aspect, the invention provides a method for identifying acompound that binds to or modulates the activity of an adenylate kinaseprotein. In general, such methods entail measuring a biological activityof an adenylate kinase protein in the presence and absence of a testcompound and identifying those compounds that alter the activity of theadenylate kinase protein.

The invention also features methods for identifying a compound thatmodulates the expression of adenylate kinase genes by measuring theexpression of the adenylate kinase sequences in the presence and absenceof the compound.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the invention are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

The present invention is based, at least in part, on the identificationof novel molecules, referred to herein as adenylate kinase nucleic acidand polypeptide molecules, which play a role in, or function in,numerous biochemical pathways associated with cellular growth and/orcellular metabolic activity. These growth and metabolic pathways aredescribed in Lodish et al. (1995) Molecular Cell Biology (ScientificAmerican Books Inc., New York, N.Y.) and Devlin (1997) Textbook ofBiochemistry with Clinical Correlations (Wiley-Liss, Inc., New York,N.Y.), the contents of which are incorporated herein by reference.

Specifically, the present invention provides isolated nucleic acidmolecules comprising nucleotide sequences encoding the adenylate kinasepolypeptide whose amino acid sequence is given in SEQ ID NO:22, or avariant or fragment of the polypeptides. A nucleotide sequence encodingan adenylate kinase polypeptide of the invention, more particularly thepolypeptide of SEQ ID NO:22, is set forth in SEQ ID NO:21.

A novel human gene, termed clone h27802 is provided. This sequence, andcomplements thereof, are referred to as “adenylate kinase” indicatingthat the gene sequences share sequence similarity to adenylate kinasegenes.

The novel h27802 adenylate kinase gene encodes an approximately 1.45 KbmRNA transcript having the corresponding cDNA set forth in SEQ ID NO:21.This transcript encodes a 258 amino acid protein (SEQ ID NO:22). Ananalysis of the full-length h27802 polypeptide predicts that theN-terminal 56 amino acids may represent a region comprising a signalpeptide. Prosite program analysis was used to predict various siteswithin the h27802 protein. See FIG. 31.

The h27802 adenylate kinase protein possesses adenylate kinase domainsequences, as shown in FIG. 34. There are three functional subdomainscommon to nucleoside monophosphate kinases: the nucleoside triphosphatebinding glycine-rich region, the nucleoside monophosphate binding site,and the lid domain that closes over the substrate upon binding (seeSchulz (1987) Cold Spring Harbor Symp. Quant. Biol. 52:429-439).

Human 27802 was aligned with two consensus amino acid sequences foradenylate kinase domains derived from hidden Markov models. For generalinformation regarding PFAM identifiers, PS prefix and PF prefix domainidentification numbers, refer to Sonnhammer et al. (1997) Protein28:405-420 and the information available at URLwww.psc.edu/general/software/packages/pfam/pfam.html. The firstadenylate kinase domain (SEQ ID NO:24) aligns with amino acids 41-120 ofSEQ ID NO:22 and the second adenylate kinase domain (SEQ ID NO:25)aligns with amino acids 201-251 of SEQ ID NO:22 (see FIG. 34).

As used herein, the term “adenylate kinase domain” includes an aminoacid sequence of about 30-200 amino acid residues in length and having abit score for the alignment of the sequence to the adenylate kinasedomain (HMM) of at least 8. Preferably, an adenylate kinase domainincludes at least about 40-150 amino acids, more preferably about 50-100amino acid residues, or about 50-80 amino acids and has a bit score forthe alignment of the sequence to the adenylate kinase domain (HMM) of atleast 16 or greater. The adenylate kinase domain (HMM) has been assignedthe PFAM Accession PF00406; see also the information available at URLpfam.wustl.edu/).

In a preferred embodiment a 27802-like polypeptide or protein has“adenylate kinase domains” or regions which include at least about30-200, more preferably about 40-100, or 50-80 amino acid residues andhas at least about 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequenceidentity with an “adenylate kinase domain,” e.g., the adenylate kinasedomain of human 27802-like (e.g., amino acid residues 41-120 and 201-251of SEQ ID NO:22).

To identify the presence of an adenylate kinase domain in a 27802-likeprotein sequence, and make the determination that a polypeptide orprotein of interest has a particular profile, the amino acid sequence ofthe protein can be searched against a database of HMMs (e.g., the Pfamdatabase, release 2.1) using the default parameters described at the URLwww.sanger.ac.uk/Software/Pfam/HMM_search. For example, the hmmsfprogram, which is available as part of the HMMER package of searchprograms, is a family specific default program for MILPAT0063 and ascore of 15 is the default threshold score for determining a hit.Alternatively, the threshold score for determining a hit can be lowered(e.g., to 8 bits). A description of the Pfam database can be found inSonhammer et al. (1997) Proteins 28(3):405-420 and a detaileddescription of HMMs can be found, for example, in Gribskov et al. (1990)Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad.Sci. USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531;and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of whichare incorporated herein by reference.

A BLASTN search using the 27802 cDNA clone of the invention as thesubject was performed. The 27802 clone shares 97% to 100% identity tothe top five search hits. These hits are 1) a partial cDNA cloneisolated from a brain glioblastoma (Accession No: AI359456) withhomology to maize adenylate kinase; 2) a partial 3′ cDNA clone isolatedfrom Jia bone marrow stroma (Accession No: A WO69362); 3) a partial cDNAclone isolated from a brain glioblastoma (Accession No: AI362274) withhomology to maize adenylate kinase; 4) a partial cDNA clone isolatedfrom a brain anaplastic oligodendroglioma (Accession No: AI826091) withhomology to maize adenylate kinase; and 5) a partial cDNA clone isolatedfrom adult heart (Accession No: CO3497).

The expression levels of 27802 were determined in various tissues byquantitative PCR (FIG. 35). The highest levels of expression of 27802were observed in artery, kidney, brain cortex and brain hypothalamus,ovary, lung (tumor), and tonsil. The expression of 27802 in a tissueindicates that modulation of the expression or activity of 27802 in thattissue may be used in the treatment of disorders involving such atissue.

In one embodiment, the adenylate kinase molecules modulate the activityof one or more proteins involved in cellular growth or differentiation,e.g., cardiac, epithelial, or neuronal cell growth or differentiation.In another embodiment, the adenylate kinase molecules of the presentinvention are capable of modulating the phosphorylation state of anucleoside mono-, di-, or triphosphate molecule or the phosphorylationstate of one or more proteins involved in cellular growth ordifferentiation, e.g., cardiac, epithelial, or neuronal cell growth ordifferentiation, as described in, for example, Lodish et al. (1995) andDevlin (1997), supra.

In addition, the adenylate kinase of the present invention are targetsof drugs described in Goodman and Gilman (1996), The PharmacologicalBasis of Therapeutics (9^(th) ed.) Hartman & Limbard Editors, thecontents of which are incorporated herein by reference. Particularly,the adenylate kinases of the invention may modulate phosphorylationactivity in tissues and cells including, but not limited to, humanbrain. In addition, expression of the gene is also observed in lymphoma.In one embodiment, the adenylate kinase sequences of the invention areused to manipulate the nucleoside mono-, di-, and triphosphate pool toalter cellular metabolic pathways, such as glycolysis andgluconeogenesis.

Adenylate kinases play an important role in the regulation of energybalance within cells and in energy-requiring biochemical processesassociated with cellular growth and development. Inhibition orover-stimulation of the activity of adenylate kinases affects thecellular equilibrium between nucleoside mono-, di-, and triphosphates,particularly AMP, ADP, and ATP, all of which are integrally involved inenergy-requiring biochemical processes associated with cellular growthand development. Disruption or modulation of this equilibrium can leadto perturbed cellular growth, which can in turn lead to cellular growthrelated-disorders. As used herein, a “cellular growth-related disorder”includes a disorder, disease, or condition characterized by aderegulation, e.g., an upregulation or a downregulation, of cellulargrowth. Cellular growth deregulation may be due to a deregulation ofcellular proliferation, cell cycle progression, cellular differentiationand/or cellular hypertrophy.

Examples of cellular growth related disorders include cardiovasculardisorders such as heart failure, hypertension, atrial fibrillation,dilated cardiomyopathy, idiopathic cardiomyopathy, or angina;proliferative disorders or differentiative disorders such as cancer,e.g., lymphoma, melanoma, prostate cancer, cervical cancer, breastcancer, colon cancer, or sarcoma.

Furthermore, adenylate kinase activity increases in cerebrospinal fluidat the acute onset of ischemic brain damage and is correlated with theseverity of the lesion (Buttner et al. (1986) J. Neurol. 233:297-303).Adenyl kinase activity also increases in cerebrospinal fluid in somebrain tumors (Ronquist et al. (1977) Lancet i: 1284-1286). Further,adenylate kinase may be expressed in damaged tissue and therefore is auseful target to measure tissue damage. Finally, deletions at 1p31 locusin many tumors is associated with hemolytic anemia (Matsuura et al.(1989) J. Biol. Chem. 264:10148-10155 and Mitelman et al. (1997) NatureGenet. 15:417-474). Accordingly, the compositions are also useful fortreatment and diagnosis related to these disorders.

The disclosed invention relates to methods and compositions for themodulation, diagnosis, and treatment of cellular proliferative and/ordifferentiative, neurological, immune, inflammatory, lymphatic,cardiovascular, respiratory, and hematological disorders.

Immune disorders include, but are not limited to, chronic inflammatorydiseases and disorders, such as Crohn's disease, reactive arthritis,including Lyme disease, insulin-dependent diabetes, organ-specificautoimmunity, including multiple sclerosis, Hashimoto's thyroiditis andGrave's disease, contact dermatitis, psoriasis, graft rejection, graftversus host disease, sarcoidosis, atopic conditions, such as asthma andallergy, including allergic rhinitis, gastrointestinal allergies,including food allergies, eosinophilia, conjunctivitis, glomerularnephritis, certain pathogen susceptibilities such as helminthic (e.g.,leishmaniasis), certain viral infections, including HIV, and bacterialinfections, including tuberculosis and lepromatous leprosy.

Disorders involving blood vessels include, but are not limited to,responses of vascular cell walls to injury, such as endothelialdysfunction and endothelial activation and intimal thickening; vasculardiseases including, but not limited to, congenital anomalies, such asarteriovenous fistula, atherosclerosis, and hypertensive vasculardisease, such as hypertension; inflammatory disease—the vasculitides,such as giant cell (temporal) arteritis, Takayasu arteritis,polyarteritis nodosa (classic), Kawasaki syndrome (mucocutaneous lymphnode syndrome), microscopic polyanglitis (microscopic polyarteritis,hypersensitivity or leukocytoclastic anglitis), Wegener granulomatosis,thromboanglitis obliterans (Buerger disease), vasculitis associated withother disorders, and infectious arteritis; Raynaud disease; aneurysmsand dissection, such as abdominal aortic aneurysms, syphilitic (luetic)aneurysms, and aortic dissection (dissecting hematoma); disorders ofveins and lymphatics, such as varicose veins, thrombophlebitis andphlebothrombosis, obstruction of superior vena cava (superior vena cavasyndrome), obstruction of inferior vena cava (inferior vena cavasyndrome), and lymphangitis and lymphedema; tumors, including benigntumors and tumor-like conditions, such as hemangioma, lymphangioma,glomus tumor (glomangioma), vascular ectasias, and bacillaryangiomatosis, and intermediate-grade (borderline low-grade malignant)tumors, such as Kaposi sarcoma and hemangloendothelioma, and malignanttumors, such as angiosarcoma and hemangiopericytoma; and pathology oftherapeutic interventions in vascular disease, such as balloonangioplasty and related techniques and vascular replacement, such ascoronary artery bypass graft surgery.

Disorders involving red cells include, but are not limited to, anemias,such as hemolytic anemias, including hereditary spherocytosis, hemolyticdisease due to erythrocyte enzyme defects: glucose-6-phosphatedehydrogenase deficiency, sickle cell disease, thalassemia syndromes,paroxysmal nocturnal hemoglobinuria, immunohemolytic anemia, andhemolytic anemia resulting from trauma to red cells; and anemias ofdiminished erythropoiesis, including megaloblastic anemias, such asanemias of vitamin B12 deficiency: pernicious anemia, and anemia offolate deficiency, iron deficiency anemia, anemia of chronic disease,aplastic anemia, pure red cell aplasia, and other forms of marrowfailure.

Hematologic disorders include but are not limited to anemias includingsickle cell and hemolytic anemia, hemophilias including types A and B,leukemias, thalassemias, spherocytosis, Von Willebrand disease, chronicgranulomatous disease, glucose-6-phosphate dehydrogenase deficiency,thrombosis, clotting factor abnormalities and deficiencies includingfactor VIII and IX deficiencies, hemarthrosis, hematemesis, hematomas,hematuria, hemochromatosis, hemoglobinuria, hemolytic-uremic syndrome,thrombocytopenias including HIV-associated thrombocytopenia, hemorrhagictelangiectasia, idiopathic thrombocytopenic purpura, thromboticmicroangiopathy, hemosiderosis.

Respiratory disorders include, but are not limited to, apnea, asthma,particularly bronchial asthma, berillium disease, bronchiectasis,bronchitis, bronchopneumonia, cystic fibrosis, diphtheria, dyspnea,emphysema, chronic obstructive pulmonary disease, allergicbronchopulmonary aspergillosis, pneumonia, acute pulmonary edema,pertussis, pharyngitis, atelectasis, Wegener's granulomatosis,Legionnaires disease, pleurisy, rheumatic fever, and sinusitis.

Preferred disorders include, but are not limited to disorders of brainand lymph node, especially lymphoma.

The disclosed invention also relates to methods and compositions for themodulation, diagnosis, and treatment of disorders involving the brainand lymph nodes.

Disorders involving the brain include, but are limited to, disordersinvolving neurons, and disorders involving glia, such as astrocytes,oligodendrocytes, ependymal cells, and microglia; cerebral edema, raisedintracranial pressure and herniation, and hydrocephalus; malformationsand developmental diseases, such as neural tube defects, forebrainanomalies, posterior fossa anomalies, and syringomyelia and hydromyelia;perinatal brain injury; cerebrovascular diseases, such as those relatedto hypoxia, ischemia, and infarction, including hypotension,hypoperfusion, and low-flow states—global cerebral ischemia and focalcerebral ischemia—infarction from obstruction of local blood supply,intracranial hemorrhage, including intracerebral (intraparenchymal)hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, andvascular malformations, hypertensive cerebrovascular disease, includinglacunar infarcts, slit hemorrhages, and hypertensive encephalopathy;infections, such as acute meningitis, including acute pyogenic(bacterial) meningitis and acute aseptic (viral) meningitis, acute focalsuppurative infections, including brain abscess, subdural empyema, andextradural abscess, chronic bacterial meningoencephalitis, includingtuberculosis and mycobacterioses, neurosyphilis, and neuroborreliosis(Lyme disease), viral meningoencephalitis, including arthropod-borne(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes simplexvirus Type 2, Varicalla-zoster virus (Herpes zoster), cytomegalovirus,poliomyelitis, rabies, and human immunodeficiency virus 1, includingHIV-1 meningoencephalitis (subacute encephalitis), vacuolar myelopathy,AIDS-associated myopathy, peripheral neuropathy, and AIDS in children,progressive multifocal leukoencephalopathy, subacute sclerosingpanencephalitis, fungal meningoencephalitis, other infectious diseasesof the nervous system; transmissible spongiform encephalopathies (priondiseases); demyelinating diseases, including multiple sclerosis,multiple sclerosis variants, acute disseminated encephalomyelitis andacute necrotizing hemorrhagic encephalomyelitis, and other diseases withdemyelination; degenerative diseases, such as degenerative diseasesaffecting the cerebral cortex, including Alzheimer disease and Pickdisease, degenerative diseases of basal ganglia and brain stem,including Parkinsonism, idiopathic Parkinson disease (paralysisagitans), progressive supranuclear palsy, corticobasal degenration,multiple system atrophy, including striatonigral degenration, Shy-Dragersyndrome, and olivopontocerebellar atrophy, and Huntington disease;spinocerebellar degenerations, including spinocerebellar ataxias,including Friedreich ataxia, and ataxia-telanglectasia, degenerativediseases affecting motor neurons, including amyotrophic lateralsclerosis (motor neuron disease), bulbospinal atrophy (Kennedysyndrome), and spinal muscular atrophy; inborn errors of metabolism,such as leukodystrophies, including Krabbe disease, metachromaticleukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, andCanavan disease, mitochondrial encephalomyopathies, including Leighdisease and other mitochondrial encephalomyopathies; toxic and acquiredmetabolic diseases, including vitamin deficiencies such as thiamine(vitamin B₁) deficiency and vitamin B₁₂ deficiency, neurologic sequelaeof metabolic disturbances, including hypoglycemia, hyperglycemia, andhepatic encephatopathy, toxic disorders, including carbon monoxide,methanol, ethanol, and radiation, including combined methotrexate andradiation-induced injury; tumors, such as gliomas, includingastrocytoma, including fibrillary (diffuse) astrocytoma and glioblastomamultiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, andbrain stem glioma, oligodendroglioma, and ependymoma and relatedparaventricular mass lesions, neuronal tumors, poorly differentiatedneoplasms, including medulloblastoma, other parenchymal tumors,including primary brain lymphoma, germ cell tumors, and pinealparenchymal tumors, meningiomas, metastatic tumors, paraneoplasticsyndromes, peripheral nerve sheath tumors, including schwannoma,neurofibroma, and malignant peripheral nerve sheath tumor (malignantschwannoma), and neurocutaneous syndromes (phakomatoses), includingneurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindaudisease.

Disorders involving T-cells include, but are not limited to,cell-mediated hypersensitivity, such as delayed type hypersensitivityand T-cell-mediated cytotoxicity, and transplant rejection; autoimmunediseases, such as systemic lupus erythematosus, Sjögren syndrome,systemic sclerosis, inflammatory myopathies, mixed connective tissuedisease, and polyarteritis nodosa and other vasculitides; immunologicdeficiency syndromes, including but not limited to, primaryimmunodeficiencies, such as thymic hypoplasia, severe combinedimmunodeficiency diseases, and AIDS; leukopenia; reactive (inflammatory)proliferations of white cells, including but not limited to,leukocytosis, acute nonspecific lymphadenitis, and chronic nonspecificlymphadenitis; neoplastic proliferations of white cells, including butnot limited to lymphoid neoplasms, such as precursor T-cell neoplasms,such as acute lymphoblastic leukemia/lymphoma, peripheral T-cell andnatural killer cell neoplasms that include peripheral T-cell lymphoma,unspecified, adult T-cell leukemia/lymphoma, mycosis fungoides andSezary syndrome, and Hodgkin disease.

In normal bone marrow, the myelocytic series (polymorphoneuclear cells)make up approximately 60% of the cellular elements, and the erythrocyticseries, 20-30%. Lymphocytes, monocytes, reticular cells, plasma cellsand megakaryocytes together constitute 10-20%. Lymphocytes make up 5-15%of normal adult marrow. In the bone marrow, cell types are add mixed sothat precursors of red blood cells (erythroblasts), macrophages(monoblasts), platelets (megakaryocytes), polymorphoneuclear leucocytes(myeloblasts), and lymphocytes (lymphoblasts) can be visible in onemicroscopic field. In addition, stem cells exist for the different celllineages, as well as a precursor stem cell for the committed progenitorcells of the different lineages. The various types of cells and stagesof each would be known to the person of ordinary skill in the art andare found, for example, on page 42 (FIG. 2-8) of Immunology,Imunopathology and Immunity, Fifth Edition, Sell et al. Simon andSchuster (1996), incorporated by reference for its teaching of celltypes found in the bone marrow. Accordingly, the invention is directedto disorders arising from these cells. These disorders include but arenot limited to the following: diseases involving hematopoeitic stemcells; committed lymphoid progenitor cells; lymphoid cells including Band T-cells; committed myeloid progenitors, including monocytes,granulocytes, and megakaryocytes; and committed erythroid progenitors.These include but are not limited to the leukemias, including B-lymphoidleukemias, T-lymphoid leukemias, undifferentiated leukemias;erythroleukemia, megakaryoblastic leukemia, monocytic; (leukemias areencompassed with and without differentiation); chronic and acutelymphoblastic leukemia, chronic and acute lymphocytic leukemia, chronicand acute myelogenous leukemia, lymphoma, myelo dysplastic syndrome,chronic and acute myeloid leukemia, myelomonocytic leukemia; chronic andacute myeloblastic leukemia, chronic and acute myelogenous leukemia,chronic and acute promyelocytic leukemia, chronic and acute myelocyticleukemia, hematologic malignancies of monocyte-macrophage lineage, suchas juvenile chronic myelogenous leukemia; secondary AML, antecedenthematological disorder; refractory anemia; aplastic anemia; reactivecutaneous angioendotheliomatosis; fibrosing disorders involving alteredexpression in dendritic cells, disorders including systemic sclerosis,E-M syndrome, epidemic toxic oil syndrome, eosinophilic fasciitislocalized forms of scleroderma, keloid, and fibrosing colonopathy;angiomatoid malignant fibrous histiocytoma; carcinoma, including primaryhead and neck squamous cell carcinoma; sarcoma, including kaposi'ssarcoma; fibroadanoma and phyllodes tumors, including mammaryfibroadenoma; stromal tumors; phyllodes tumors, including histiocytoma;erythroblastosis; neurofibromatosis; diseases of the vascularendothelium; demyelinating, particularly in old lesions; gliosis,vasogenic edema, vascular disease, Alzheimer's and Parkinson's disease;T-cell lymphomas; B-cell lymphomas.

Disorders involving B-cells include, but are not limited to precursorB-cell neoplasms, such as lymphoblastic leukemia/lymphoma. PeripheralB-cell neoplasms include, but are not limited to, chronic lymphocyticleukemia/small lymphocytic lymphoma, follicular lymphoma, diffuse largeB-cell lymphoma, Burkitt lymphoma, plasma cell neoplasms, multiplemyeloma, and related entities, lymphoplasmacytic lymphoma(Waldenstr{overscore (o)}m macroglobulinemia), mantle cell lymphoma,marginal zone lymphoma (MALToma), and hairy cell leukemia.

Disorders involving precursor T-cell neoplasms include precursor Tlymphoblastic leukemia/lymphoma. Disorders involving peripheral T-celland natural killer cell neoplasms include T-cell chronic lymphocyticleukemia, large granular lymphocytic leukemia, mycosis fungoides andSézary syndrome, peripheral T-cell lymphoma, unspecified,angioimmunoblastic T-cell lymphoma, angiocentric lymphoma (NK/T-celllymphoma^(4a)), intestinal T-cell lymphoma, adult T-cellleukemia/lymphoma, and anaplastic large cell lymphoma.

The adenylate kinase sequences of the invention are members of a familyof molecules having conserved functional features. The term “family”when referring to the proteins and nucleic acid molecules of theinvention is intended to mean two or more proteins or nucleic acidmolecules having sufficient amino acid or nucleotide sequence identityas defined herein. Such family members can be naturally occurring andcan be from either the same or different species. For example, a familycan contain a first protein of murine origin and a homologue of thatprotein of human origin, as well as a second, distinct protein of humanorigin and a murine homologue of that protein. Members of a family mayalso have common functional characteristics.

Preferred adenylate kinase polypeptides of the present invention have anamino acid sequence sufficiently identical to the amino acid sequence ofSEQ ID NO:22. The term “sufficiently identical” is used herein to referto a first amino acid or nucleotide sequence that contains a sufficientor minimum number of identical or equivalent (e.g., with a similar sidechain) amino acid residues or nucleotides to a second amino acid ornucleotide sequence such that the first and second amino acid ornucleotide sequences have a common structural domain and/or commonfunctional activity. For example, amino acid or nucleotide sequencesthat contain a common structural domain having at least about 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity are definedherein as sufficiently identical.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, 100% of the length of thereference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (1970)J. Mol. Biol. 48:444-453 algorithm which has been incorporated into theGAP program in the GCG software package (available at the URLwww.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and agap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3,4, 5, or 6. In yet another preferred embodiment, the percent identitybetween two nucleotide sequences is determined using the GAP program inthe GCG software package (available at http://www.gcg.com), using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and alength weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set ofparameters (and the one that should be used if the practitioner isuncertain about what parameters should be applied to determine if amolecule is within a sequence identity or homology limitation of theinvention) is using a Blossum 62 scoring matrix with a gap open penaltyof 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of E. Meyers and W. Miller (1989)CABIOS 4:11-17 which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to 27802 nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to 27802 protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al. (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See information at the URL www.ncbi.nlm.nih.gov.

Accordingly, another embodiment of the invention features isolatedadenylate kinase proteins and polypeptides having an adenylate kinaseprotein activity. As used interchangeably herein, an “adenylate kinaseprotein activity”, “biological activity of an adenylate kinase protein”,or “functional activity of an adenylate kinase protein” refers to anactivity exerted by an adenylate kinase protein, polypeptide, or nucleicacid molecule on an adenylate kinase responsive cell as determined invivo, or in vitro, according to standard assay techniques. An adenylatekinase activity can be a direct activity, such as an association with oran enzymatic activity on a second protein, or an indirect activity, suchas a cellular activity mediated by interaction of the adenylate kinaseprotein with a second protein. In a preferred embodiment, an adenylatekinase activity includes at least one or more of the followingactivities: (1) modulating (stimulating and/or enhancing or inhibiting)cellular proliferation, differentiation, and/or function, particularlyin cells in which the sequences are expressed, for example, cells of thelymph node, including Th1, Th2, T cells, natural killer T cells,lymphocytes, leukocytes, etc., and brain, such as glial cells andneurons; (2) modulating a target cell's energy balance, particularly theratio between AMP and ATP; (3) modulating the glycolytic pathway; (4)modulating the gluconeogenesis pathway; (4) modulating cell growth; (5)modulating the entry of cells into mitosis; (6) modulating cellulardifferentiation; (7) modulating cell death; and (8) modulating an immuneresponse.

An “isolated” or “purified” adenylate kinase nucleic acid molecule orprotein, or biologically active portion thereof, is substantially freeof other cellular material, or culture medium when produced byrecombinant techniques, or substantially free of chemical precursors orother chemicals when chemically synthesized. Preferably, an “isolated”nucleic acid is free of sequences (preferably protein encodingsequences) that naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For purposes of theinvention, “isolated” when used to refer to nucleic acid moleculesexcludes isolated chromosomes. For example, in various embodiments, theisolated adenylate kinase nucleic acid molecule can contain less thanabout 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotidesequences that naturally flank the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived. An adenylate kinaseprotein that is substantially free of cellular material includespreparations of adenylate kinase protein having less than about 30%,20%, 10%, or 5% (by dry weight) of non-adenylate kinase protein (alsoreferred to herein as a “contaminating protein”). When the adenylatekinase protein or biologically active portion thereof is recombinantlyproduced, preferably, culture medium represents less than about 30%,20%, 10%, or 5% of the volume of the protein preparation. When adenylatekinase protein is produced by chemical synthesis, preferably the proteinpreparations have less than about 30%, 20%, 10%, or 5% (by dry weight)of chemical precursors or non-adenylate kinase chemicals.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculescomprising nucleotide sequences encoding adenylate kinase proteins andpolypeptides or biologically active portions thereof, as well as nucleicacid molecules sufficient for use as hybridization probes to identifyadenylate kinase-encoding nucleic acids (e.g., adenylate kinase mRNA)and fragments for use as PCR primers for the amplification or mutationof adenylate kinase nucleic acid molecules. As used herein, the term“nucleic acid molecule” is intended to include DNA molecules (e.g., cDNAor genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

Nucleotide sequences encoding the adenylate kinase proteins of thepresent invention include sequences set forth in SEQ ID NO:21 andcomplements thereof. By “complement” is intended a nucleotide sequencethat is sufficiently complementary to a given nucleotide sequence suchthat it can hybridize to the given nucleotide sequence to thereby form astable duplex. The corresponding amino acid sequence for the adenylatekinase protein encoded by these nucleotide sequences is set forth in SEQID NO:22.

Nucleic acid molecules that are fragments of these adenylate kinasenucleotide sequences are also encompassed by the present invention. By“fragment” is intended a portion of the nucleotide sequence encoding anadenylate kinase protein. A fragment of an adenylate kinase nucleotidesequence may encode a biologically active portion of an adenylate kinaseprotein, or it may be a fragment that can be used as a hybridizationprobe or PCR primer using methods disclosed below. A biologically activeportion of an adenylate kinase protein can be prepared by isolating aportion of one of the adenylate kinase nucleotide sequences of theinvention, expressing the encoded portion of the adenylate kinaseprotein (e.g., by recombinant expression in vitro), and assessing theactivity of the encoded portion of the adenylate kinase protein. Nucleicacid molecules that are fragments of an adenylate kinase nucleotidesequence comprise at least 15, 20, 50, 75, 100, 200, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400 nucleotides, or up to the number ofnucleotides present in a full-length adenylate kinase nucleotidesequence disclosed herein (for example, 1452 nucleotides for SEQ IDNO:21) depending upon the intended use.

Alternatively, a nucleic acid molecule that is a fragment of an27802-like nucleotide sequence of the present invention comprises anucleotide sequence consisting of at least 5, 10, 15, 20, 25, 30, 35, or40 contiguous nucleotides of nucleotides 215-370, or nucleotides 843-941of SEQ ID NO:21. A fragment of a nucleotide sequence of the presentinvention comprises a nucleotide sequence consisting of nucleotides215-300, 300-370, 843-900, 900-941 of SEQ ID NO:21.

It is understood that isolated fragments include any contiguous sequencenot disclosed prior to the invention as well as sequences that aresubstantially the same and which are not disclosed. Accordingly, if anisolated fragment is disclosed prior to the present invention, thatfragment is not intended to be encompassed by the invention. When asequence is not disclosed prior to the present invention, an isolatednucleic acid fragment is at least about 5-10, 10-15, 15-20, 20-25,25-30, 30-35, 35-40, 40-45, 45-50, 50-75, 75-100 or more contiguousnucleotides. Other regions of the nucleotide sequence may comprisefragments of various sizes, depending upon potential homology withpreviously disclosed sequences.

A fragment of an adenylate kinase nucleotide sequence that encodes abiologically active portion of an adenylate kinase protein of theinvention will encode at least 15, 25, 30, 50, 75, 100, 125, 150, 175,200, or 225 contiguous amino acids, or up to the total number of aminoacids present in a full-length adenylate kinase protein of the invention(for example, 258 amino acids for SEQ ID NO:22). A nucleic acid moleculethat is a fragment of an 27802-like nucleotide sequence of the presentinvention comprises a nucleotide sequence encoding at least 15, 20, 25,30, 35, or 40 contiguous amino acids of amino acids 1-51, or 209-241 ofSEQ ID NO:22. A fragment of a nucleotide sequence of the presentinvention comprises a nucleotide sequence encoding amino acids 1-25,25-51, 209-241 of SEQ ID NO:22.

Fragments of an adenylate kinase nucleotide sequence that are useful ashybridization probes for PCR primers generally need not encode abiologically active portion of an adenylate kinase protein.

Nucleic acid molecules that are variants of the adenylate kinasenucleotide sequences disclosed herein are also encompassed by thepresent invention. “Variants” of the adenylate kinase nucleotidesequences include those sequences that encode the adenylate kinaseproteins disclosed herein but that differ conservatively because of thedegeneracy of the genetic code. These naturally occurring allelicvariants can be identified with the use of well-known molecular biologytechniques, such as polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant nucleotide sequences also includesynthetically derived nucleotide sequences that have been generated, forexample, by using site-directed mutagenesis but which still encode theadenylate kinase proteins disclosed in the present invention asdiscussed below. Generally, nucleotide sequence variants of theinvention with have at least 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequencedisclosed herein. A variant adenylate kinase nucleotide sequence willencode an adenylate kinase protein that has an amino acid sequencehaving at least 45%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequenceof an adenylate kinase protein disclosed herein. Such variants retainthe functional activity (e.g., the adenylate kinase activity) of thepolypeptide set forth in SEQ ID NO:22.

In addition to the adenylate kinase nucleotide sequence shown in SEQ IDNO:21, it will be appreciated by those skilled in the art that DNAsequence polymorphisms that lead to changes in the amino acid sequencesof adenylate kinase proteins may exist within a population (e.g., thehuman population). Such genetic polymorphism in an adenylate kinase genemay exist among individuals within a population due to natural allelicvariation. An allele is one of a group of genes that occur alternativelyat a given genetic locus. As used herein, the terms “gene” and“recombinant gene” refer to nucleic acid molecules comprising an openreading frame encoding an adenylate kinase protein, preferably amammalian adenylate kinase protein. As used herein, the phrase “allelicvariant” refers to a nucleotide sequence that occurs at an adenylatekinase locus or to a polypeptide encoded by the nucleotide sequence.Such natural allelic variations can typically result in 1-5% variance inthe nucleotide sequence of the adenylate kinase gene. Any and all suchnucleotide variations and resulting amino acid polymorphisms orvariations in an adenylate kinase sequence that are the result ofnatural allelic variation and that do not alter the functional activityof adenylate kinase proteins are intended to be within the scope of theinvention.

Moreover, nucleic acid molecules encoding adenylate kinase proteins fromother species (adenylate kinase homologs), which have a nucleotidesequence differing from that of the adenylate kinase sequence disclosedherein, are intended to be within the scope of the invention. Forexample, nucleic acid molecules corresponding to natural allelicvariants and homologs of the human adenylate kinase cDNA of theinvention can be isolated based on their identity to the human adenylatekinase nucleic acid disclosed herein using the human cDNA, or a portionthereof, as a hybridization probe according to standard hybridizationtechniques under stringent hybridization conditions as disclosed below.

In addition to naturally-occurring allelic variants of the adenylatekinase sequences that may exist in the population, the skilled artisanwill further appreciate that changes can be introduced by mutation intothe nucleotide sequences of the invention thereby leading to changes inthe amino acid sequence of the encoded adenylate kinase proteins,without altering the biological activity of the adenylate kinaseproteins. Thus, an isolated nucleic acid molecule encoding an adenylatekinase protein having a sequence that differs from that of SEQ ID NO:22can be created by introducing one or more nucleotide substitutions,additions, or deletions into the corresponding nucleotide sequencedisclosed herein, such that one or more amino acid substitutions,additions or deletions are introduced into the encoded protein.Mutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Such variantnucleotide sequences are also encompassed by the present invention.

For example, preferably, conservative amino acid substitutions may bemade at one or more predicted, preferably nonessential amino acidresidues. A “nonessential” amino acid residue is a residue that can bealtered from the wild-type sequence of an adenylate kinase protein(e.g., the sequence of SEQ ID NO:22) without altering the biologicalactivity, whereas an “essential” amino acid residue is required forbiological activity. A “conservative amino acid substitution” is one inwhich the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. These families includeamino acids with basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Such substitutions would not be made for conserved aminoacid residues, or for amino acid residues residing within a conservedmotif, such as the adenylate kinase domain sequence of SEQ ID NO:22 (seeFIG. 30), where such residues are essential for protein activity.

Alternatively, variant adenylate kinase nucleotide sequences can be madeby introducing mutations randomly along all or part of an adenylatekinase coding sequence, such as by saturation mutagenesis, and theresultant mutants can be screened for, adenylate kinase biologicalactivity to identify mutants that retain activity. Followingmutagenesis, the encoded protein can be expressed recombinantly, and theactivity of the protein can be determined using standard assaytechniques.

Thus the nucleotide sequence of the invention includes the sequencedisclosed herein as well as fragments and variants thereof. Theadenylate kinase nucleotide sequence of the invention, and fragments andvariants thereof, can be used as probes and/or primers to identifyand/or clone adenylate kinase homologs in other cell types, e.g., fromother tissues, as well as adenylate kinase homologs from other mammals.Such probes can be used to detect transcripts or genomic sequencesencoding the same or identical proteins. These probes can be used aspart of a diagnostic test kit for identifying cells or tissues thatmisexpress an adenylate kinase protein, such as by measuring levels ofan adenylate kinase-encoding nucleic acid in a sample of cells from asubject, e.g., detecting adenylate kinase mRNA levels or determiningwhether a genomic adenylate kinase gene has been mutated or deleted.

In this manner, methods such as PCR, hybridization, and the like can beused to identify such sequences having substantial identity to thesequences of the invention. See, for example, Sambrook et al. (1989)Molecular Cloning: Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.) and Innis, et al. (1990) PCRProtocols: A Guide to Methods and Applications (Academic Press, NY).Adenylate kinase nucleotide sequences isolated based on their sequenceidentity to the adenylate kinase nucleotide sequence set forth herein orto fragments and variants thereof are encompassed by the presentinvention.

In a hybridization method, all or part of a known adenylate kinasenucleotide sequence can be used to screen cDNA or genomic libraries.Methods for construction of such cDNA and genomic libraries aregenerally known in the art and are disclosed in Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.). The so-called hybridization probesmay be genomic DNA fragments, cDNA fragments, RNA fragments, or otheroligonucleotides, and may be labeled with a detectable group such as³²P, or any other detectable marker, such as other radioisotopes, afluorescent compound, an enzyme, or an enzyme co-factor. Probes forhybridization can be made by labeling synthetic oligonucleotides basedon the known adenylate kinase nucleotide sequence disclosed herein.Degenerate primers designed on the basis of conserved nucleotides oramino acid residues in a known adenylate kinase nucleotide sequence orencoded amino acid sequence can additionally be used. The probetypically comprises a region of nucleotide sequence that hybridizesunder stringent conditions to at least about 12, preferably about 25,more preferably about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or400 consecutive nucleotides of an adenylate kinase nucleotide sequenceof the invention or a fragment or variant thereof. Preparation of probesfor hybridization is generally known in the art and is disclosed inSambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.,Cold Spring Harbor Laboratory Press, Plainview, N.Y.), hereinincorporated by reference.

For example, in one embodiment, a previously unidentified adenylatekinase nucleic acid molecule hybridizes under stringent conditions to aprobe that is a nucleic acid molecule comprising the adenylate kinasenucleotide sequence of the invention or a fragment thereof. In anotherembodiment, the previously unknown adenylate kinase nucleic acidmolecule is at least 300, 325, 350, 375, 400, 425, 450, 500, 550, 600,650, 700, 800, 900, 1000, 2,000, 3,000, 4,000 or 5,000 nucleotides inlength and hybridizes under stringent conditions to a probe that is anucleic acid molecule comprising the adenylate kinase nucleotidesequence disclosed herein or a fragment thereof.

Accordingly, in another embodiment, an isolated previously unknownadenylate kinase nucleic acid molecule of the invention is at least 300,325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000,1,100, 1,200, 1,300, or 1,400 nucleotides in length and hybridizes understringent conditions to a probe that is a nucleic acid moleculecomprising the nucleotide sequence of the invention, preferably thecoding sequence set forth in SEQ ID NO:21 or a complement, fragment, orvariant thereof.

As used herein, the term “hybridizes under stringent conditions”describes conditions for hybridization and washing. Stringent conditionsare known to those skilled in the art and can be found in CurrentProtocols in Molecular Biology John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. Aqueous and nonaqueous methods are described in thatreference and either can be used. A preferred, example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50° C. Another example of stringent hybridizationconditions are hybridization in 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at55° C. A further example of stringent hybridization conditions arehybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.Preferably, stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 65° C. Particularly preferredstringency conditions (and the conditions that should be used if thepractitioner is uncertain about what conditions should be applied todetermine if a molecule is within a hybridization limitation of theinvention) are 0.5M Sodium Phosphate, 7% SDS at 65° C., followed by oneor more washes at 0.2×SSC, 1% SDS at 65° C. Preferably, an isolatednucleic acid molecule of the invention that hybridizes under stringentconditions to the sequence of SEQ ID NO:21, or SEQ ID NO:23, correspondsto a naturally-occurring nucleic acid molecule.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein).

Thus, in addition to the adenylate kinase nucleotide sequences disclosedherein and fragments and variants thereof, the isolated nucleic acidmolecules of the invention also encompass homologous DNA sequencesidentified and isolated from other cells and/or organisms byhybridization with entire or partial sequences obtained from theadenylate kinase nucleotide sequences disclosed herein or variants andfragments thereof.

The present invention also encompasses antisense nucleic acid molecules,i.e., molecules that are complementary to a sense nucleic acid encodinga protein, e.g., complementary to the coding strand of a double-strandedcDNA molecule, or complementary to an mRNA sequence. Accordingly, anantisense nucleic acid can hydrogen bond to a sense nucleic acid. Theantisense nucleic acid can be complementary to an entire adenylatekinase coding strand, or to only a portion thereof, e.g., all or part ofthe protein coding region (or open reading frame). An antisense nucleicacid molecule can be antisense to a noncoding region of the codingstrand of a nucleotide sequence encoding an adenylate kinase protein.The noncoding regions are the 5′ and 3′ sequences that flank the codingregion and are not translated into amino acids.

Given the coding-strand sequence encoding an adenylate kinase proteindisclosed herein (SEQ ID NO:21), antisense nucleic acids of theinvention can be designed according to the rules of Watson and Crickbase pairing. The antisense nucleic acid molecule can be complementaryto the entire coding region of adenylate kinase mRNA, but morepreferably is an oligonucleotide that is antisense to only a portion ofthe coding or noncoding region of adenylate kinase mRNA. For example,the antisense oligonucleotide can be complementary to the regionsurrounding the translation start site of adenylate kinase mRNA. Anantisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45, or 50 nucleotides in length. An antisense nucleic acidof the invention can be constructed using chemical synthesis andenzymatic ligation procedures known in the art.

For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, including, but not limited to, for example e.g., phosphorothioatederivatives and acridine substituted nucleotides. Alternatively, theantisense nucleic acid can be produced biologically using an expressionvector into which a nucleic acid has been subcloned in an antisenseorientation (i.e., RNA transcribed from the inserted nucleic acid willbe of an antisense orientation to a target nucleic acid of interest,described further in the following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding an adenylatekinase protein to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. An example of a route ofadministration of antisense nucleic acid molecules of the inventionincludes direct injection at a tissue site. Alternatively, antisensenucleic acid molecules can be modified to target selected cells and thenadministered systemically. For example, antisense molecules can belinked to peptides or antibodies to form a complex that specificallybinds to receptors or antigens expressed on a selected cell surface. Theantisense nucleic acid molecules can also be delivered to cells usingthe vectors described herein. To achieve sufficient intracellularconcentrations of the antisense molecules, vector constructs in whichthe antisense nucleic acid molecule is placed under the control of astrong pol II or pol III promoter are preferred.

An antisense nucleic acid molecule of the invention can be an α-anomericnucleic acid molecule. An α-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

The invention also encompasses ribozymes, which are catalytic RNAmolecules with ribonuclease activity that are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Ribozymes (e.g., hammerhead ribozymes (describedin Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used tocatalytically cleave adenylate kinase mRNA transcripts to therebyinhibit translation of adenylate kinase mRNA. A ribozyme havingspecificity for an adenylate kinase-encoding nucleic acid can bedesigned based upon the nucleotide sequence of an adenylate kinase cDNAdisclosed herein (e.g., SEQ ID NO:21). See, e.g., Cech et al., U.S. Pat.No. 4,987,071; and Cech et al., U.S. Pat. No. 5,116,742. Alternatively,adenylate kinase mRNA can be used to select a catalytic RNA having aspecific ribonuclease activity from a pool of RNA molecules. See, e.g.,Bartel and Szostak (1993) Science 261:1411-1418.

The invention also encompasses nucleic acid molecules that form triplehelical structures. For example, adenylate kinase gene expression can beinhibited by targeting nucleotide sequences complementary to theregulatory region of the adenylate kinase protein (e.g., the adenylatekinase promoter and/or enhancers) to form triple helical structures thatprevent transcription of the adenylate kinase gene in target cells. Seegenerally Helene (1991) Anticancer Drug Des. 6(6):569; Helene (1992)Ann. N.Y. Acad. Sci. 660:27; and Maher (1992) Bioassays 14(12):807.

In preferred embodiments, the nucleic acid molecules of the inventioncan be modified at the base moiety, sugar moiety, or phosphate backboneto improve, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal. (1996) Biooganic & Medicinal Chemistry 4:5). As used herein, theterms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics,e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid-phase peptide synthesis protocols as described in Hyrupet al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci.USA 93:14670.

PNAs of an adenylate kinase molecule can be used in therapeutic anddiagnostic applications. For example, PNAs can be used as antisense orantigene agents for sequence-specific modulation of gene expression by,e.g., inducing transcription or translation arrest or inhibitingreplication. PNAs of the invention can also be used, e.g., in theanalysis of single base pair mutations in a gene by, e.g., PNA-directedPCR clamping; as artificial restriction enzymes when used in combinationwith other enzymes, e.g., S1 nucleases (Hyrup (1996), supra; or asprobes or primers for DNA sequence and hybridization (Hyrup (1996),supra; Perry-O'Keefe et al. (1996), supra).

In another embodiment, PNAs of an adenylate kinase molecule can bemodified, e.g., to enhance their stability, specificity, or cellularuptake, by attaching lipophilic or other helper groups to PNA, by theformation of PNA-DNA chimeras, or by the use of liposomes or othertechniques of drug delivery known in the art. The synthesis of PNA-DNAchimeras can be performed as described in Hyrup (1996), supra; Finn etal. (1996) Nucleic Acids Res. 24(17):3357-63; Mag et al. (1989) NucleicAcids Res. 17:5973; and Peterson et al. (1975) Bioorganic Med. Chem.Lett. 5:1119.

II. Isolated Adenylate Kinase Proteins and Anti-Adenylate KinaseAntibodies

Adenylate kinase proteins are also encompassed within the presentinvention. By “adenylate kinase protein” is intended a protein havingthe amino acid sequence set forth in SEQ ID NO:22, as well as fragments,biologically active portions, and variants thereof.

“Fragments” or “biologically active portions” include polypeptidefragments suitable for use as immunogens to raise anti-adenylate kinaseantibodies. Fragments include peptides comprising amino acid sequencessufficiently identical to or derived from the amino acid sequence of anadenylate kinase protein of the invention and exhibiting at least oneactivity of an adenylate kinase protein, but which include fewer aminoacids than the full-length (SEQ ID NO:22) adenylate kinase proteindisclosed herein. Typically, biologically active portions comprise adomain or motif with at least one activity of the adenylate kinaseprotein. A biologically active portion of an adenylate kinase proteincan be a polypeptide that is, for example, 10-15, 15-20, 20-25, 25-30,30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 70, 80, 90, 100 or more aminoacids in length. Such biologically active portions can be prepared byrecombinant techniques and evaluated for one or more of the functionalactivities of a native adenylate kinase protein. As used here, afragment comprises at least 5 contiguous amino acids of SEQ ID NO:22.The invention encompasses other fragments, however, such as any fragmentin the protein greater than 5 amino acids, depending upon the intendeduse.

By “variants” is intended proteins or polypeptides having an amino acidsequence that is at least about 45%, 55%, 65%, preferably about 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence of SEQ ID NO:22. Variants alsoinclude polypeptides encoded by a nucleic acid molecule that hybridizesto the nucleic acid molecule of SEQ ID NO:21, or a complement thereof,under stringent conditions. In another embodiment, a variant of anisolated polypeptide of the present invention differs, by at least 1,but less than 5, 10, 20, 50, or 100 amino acid residues from thesequence shown in SEQ ID NO:22. If alignment is needed for thiscomparison the sequences should be aligned for maximum identity.“Looped” out sequences from deletions or insertions, or mismatches, areconsidered differences. Such variants generally retain the functionalactivity of the 27802-like proteins of the invention. Variants includepolypeptides that differ in amino acid sequence due to natural allelicvariation or mutagenesis.

The invention also provides adenylate kinase chimeric or fusionproteins. In the case where an expression cassette contains two proteincoding regions joined in a contiguous manner in the same reading frame,the encoded polypeptide is herein defined as a “heterologouspolypeptide” or a “chimeric polypeptide” or a “fusion polypeptide”. Asused herein, an adenylate kinase “heterologous protein” or “chimericprotein” or “fusion protein” comprises an adenylate kinase polypeptideoperably linked to a non-adenylate kinase polypeptide. An “adenylatekinase polypeptide” refers to a polypeptide having an amino acidsequence corresponding to an adenylate kinase protein, whereas a“non-adenylate kinase polypeptide” refers to a polypeptide having anamino acid sequence corresponding to a protein that is not substantiallyidentical to the adenylate kinase protein, e.g., a protein that isdifferent from the adenylate kinase protein and which is derived fromthe same or a different organism. Within an adenylate kinase fusionprotein, the adenylate kinase polypeptide can correspond to all or aportion of an adenylate kinase protein, preferably at least onebiologically active portion of an adenylate kinase protein. Within thefusion protein, the term “operably linked” is intended to indicate thatthe adenylate kinase polypeptide and the non-adenylate kinasepolypeptide are fused in-frame to each other. The non-adenylate kinasepolypeptide can be fused to the N-terminus or C-terminus of theadenylate kinase polypeptide.

One useful fusion protein is a GST-adenylate kinase fusion protein inwhich the adenylate kinase sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant adenylate kinase proteins.

In yet another embodiment, the fusion protein is an adenylatekinase-immunoglobulin fusion protein in which all or part of anadenylate kinase protein is fused to sequences derived from a member ofthe immunoglobulin protein family. The adenylate kinase-immunoglobulinfusion proteins of the invention can be incorporated into pharmaceuticalcompositions and administered to a subject to inhibit an interactionbetween an adenylate kinase ligand and an adenylate kinase protein onthe surface of a cell, thereby suppressing adenylate kinase-mediatedsignal transduction in vivo. The adenylate kinase-immunoglobulin fusionproteins can be used to affect the bioavailability of an adenylatekinase cognate ligand. Inhibition of the adenylate kinaseligand/adenylate kinase interaction may be useful therapeutically, bothfor treating proliferative and differentiative disorders and formodulating (e.g., promoting or inhibiting) cell survival. Moreover, theadenylate kinase-immunoglobulin fusion proteins of the invention can beused as immunogens to produce anti-adenylate kinase antibodies in asubject, to purify adenylate kinase ligands, and in screening assays toidentify molecules that inhibit the interaction of an adenylate kinaseprotein with an adenylate kinase ligand.

Preferably, an adenylate kinase chimeric or fusion protein of theinvention is produced by standard recombinant DNA techniques. Forexample, DNA fragments coding for the different polypeptide sequencesmay be ligated together in-frame, or the fusion gene can be synthesized,such as with automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primersthat give rise to complementary overhangs between two consecutive genefragments, which can subsequently be annealed and reamplified togenerate a chimeric gene sequence (see, e.g., Ausubel et al., eds.(1995) Current Protocols in Molecular Biology) (Greene Publishing andWiley-Interscience, NY). Moreover, an adenylate kinase-encoding nucleicacid can be cloned into a commercially available expression vector suchthat it is linked in-frame to an existing fusion moiety.

Variants of the adenylate kinase proteins can function as eitheradenylate kinase agonists (mimetics) or as adenylate kinase antagonists.Variants of the adenylate kinase protein can be generated bymutagenesis, e.g., discrete point mutation or truncation of theadenylate kinase protein. An agonist of the adenylate kinase protein canretain substantially the same, or a subset, of the biological activitiesof the naturally occurring form of the adenylate kinase protein. Anantagonist of the adenylate kinase protein can inhibit one or more ofthe activities of the naturally occurring form of the adenylate kinaseprotein by, for example, competitively binding to a downstream orupstream member of a cellular signaling cascade that includes theadenylate kinase protein. Thus, specific biological effects can beelicited by treatment with a variant of limited function. Treatment of asubject with a variant having a subset of the biological activities ofthe naturally occurring form of the protein can have fewer side effectsin a subject relative to treatment with the naturally occurring form ofthe adenylate kinase proteins.

Variants of the adenylate kinase proteins can function as eitheradenylate kinase agonists (mimetics) or as adenylate kinase antagonists.Variants of the adenylate kinase protein can be generated bymutagenesis, e.g. discrete point mutation or truncation of the adenylatekinase protein. An agonist of the adenylate kinase protein can retainsubstantially the same, or a subset, of the biological activities of thenaturally occurring form of the adenylate kinase protein. An antagonistof the adenylate kinase protein can inhibit one or more of theactivities of the naturally occurring form of the adenylate kinaseprotein by, for example, competitively binding to a downstream orupstream member of a cellular signaling cascade that includes theadenylate kinase protein. Thus, specific biological effects can beelicited by treatment with a variant of limited function. Treatment of asubject with a variant having a subset of the biological activities ofthe naturally occurring form of the protein can have fewer side effectsin a subject relative to treatment with the naturally occurring form ofthe adenylate kinase proteins.

Variants of an adenylate kinase protein that function as eitheradenylate kinase agonists or as adenylate kinase antagonists can beidentified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of an adenylate kinase protein for adenylate kinaseprotein agonist or antagonist activity. In one embodiment, a variegatedlibrary of adenylate kinase variants is generated by combinatorialmutagenesis at the nucleic acid level and is encoded by a variegatedgene library. A variegated library of adenylate kinase variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential adenylate kinase sequences is expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display) containing the set of adenylate kinasesequences therein. There are a variety of methods that can be used toproduce libraries of potential adenylate kinase variants from adegenerate oligonucleotide sequence. Chemical synthesis of a degenerategene sequence can be performed in an automatic DNA synthesizer, and thesynthetic gene then ligated into an appropriate expression vector. Useof a degenerate set of genes allows for the provision, in one mixture,of all of the sequences encoding the desired set of potential adenylatekinase sequences. Methods for synthesizing degenerate oligonucleotidesare known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakuraet al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477).

In addition, libraries of fragments of an adenylate kinase proteincoding sequence can be used to generate a variegated population ofadenylate kinase fragments for screening and subsequent selection ofvariants of an adenylate kinase protein. In one embodiment, a library ofcoding sequence fragments can be generated by treating a double-strandedPCR fragment of an adenylate kinase coding sequence with a nucleaseunder conditions wherein nicking occurs only about once per molecule,denaturing the double-stranded DNA, renaturing the DNA to formdouble-stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single-stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method,one can derive an expression library that encodes N-terminal andinternal fragments of various sizes of the adenylate kinase protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of adenylate kinase proteins.The most widely used techniques, which are amenable to high throughputanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquethat enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify adenylatekinase variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).

An isolated adenylate kinase polypeptide of the invention can be used asan immunogen to generate antibodies that bind adenylate kinase proteinsusing standard techniques for polyclonal and monoclonal antibodypreparation. The full-length adenylate kinase protein can be used or,alternatively, the invention provides antigenic peptide fragments ofadenylate kinase proteins for use as immunogens. The antigenic peptideof an adenylate kinase protein comprises at least 8, preferably 10-15,15-20, 20-25, or 30 or more amino acid residues of the amino acidsequence shown in SEQ ID NO:22 and encompasses an epitope of anadenylate kinase protein such that an antibody raised against thepeptide forms a specific immune complex with the adenylate kinaseprotein. Preferred epitopes encompassed by the antigenic peptide areregions of a adenylate kinase protein that are located on the surface ofthe protein, e.g., hydrophilic regions.

Accordingly, another aspect of the invention pertains to anti-adenylatekinase polyclonal and monoclonal antibodies that bind an adenylatekinase protein. Polyclonal anti-adenylate kinase antibodies can beprepared by immunizing a suitable subject (e.g., rabbit, goat, mouse, orother mammal) with an adenylate kinase immunogen. The anti-adenylatekinase antibody titer in the immunized subject can be monitored overtime by standard techniques, such as with an enzyme linked immunosorbentassay (ELISA) using immobilized adenylate kinase protein. At anappropriate time after immunization, e.g., when the anti-adenylatekinase antibody titers are highest, antibody-producing cells can beobtained from the subject and used to prepare monoclonal antibodies bystandard techniques, such as the hybridoma technique originallydescribed by Kohler and Milstein (1975) Nature 256:495-497, the human Bcell hybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), theEBV-hybridoma technique (Cole et al. (1985) in Monoclonal Antibodies andCancer Therapy, ed. Reisfeld and Sell (Alan R. Liss, Inc., New York,N.Y.), pp. 77-96) or trioma techniques. The technology for producinghybridomas is well known (see generally Coligan et al., eds. (1994)Current Protocols in Immunology (John Wiley & Sons, Inc., New York,N.Y.); Galfre et al. (1977) Nature 266:55052; Kenneth (1980) inMonoclonal Antibodies: A New Dimension In Biological Analyses (PlenumPublishing Corp., NY; and Lerner (1981) Yale J. Biol. Med., 54:387-402).

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-adenylate kinase antibody can be identified and isolatedby screening a recombinant combinatorial immunoglobulin library (e.g.,an antibody phage display library) with an adenylate kinase protein tothereby isolate immunoglobulin library members that bind the adenylatekinase protein. Kits for generating and screening phage displaylibraries are commercially available (e.g., the Pharmacia RecombinantPhage Antibody System, Catalog No. 27-9400-01; and the StratageneSurfZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examplesof methods and reagents particularly amenable for use in generating andscreening antibody display library can be found in, for example, U.S.Pat. No. 5,223,409; PCT Publication Nos. WO 92/18619; WO 91/17271; WO92/20791; WO 92/15679; 93/01288; WO 92/01047; 92/09690; and 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffiths et al. (1993) EMBO J. 12:725-734.

Additionally, recombinant anti-adenylate kinase antibodies, such aschimeric and humanized monoclonal antibodies, comprising both human andnonhuman portions, which can be made using standard recombinant DNAtechniques, are within the scope of the invention. Such chimeric andhumanized monoclonal antibodies can be produced by recombinant DNAtechniques known in the art, for example using methods described in PCTPublication Nos. WO 86101533 and WO 87/02671; European PatentApplication Nos. 184,187, 171, 496, 125,023, and 173,494; U.S. Pat. Nos.4,816,567 and 5,225,539; European Patent Application 125,023; Better etal. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad.Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sunet al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al.(1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449;Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison (1985)Science 229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; Jones etal. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534;and Beidler et al. (1988) J. Immunol. 141:4053-4060.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice that are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. See, for example, Lonberg and Huszar (1995)Int. Rev. Immunol. 13:65-93); and U.S. Pat. Nos. 5,625,126; 5,633,425;5,569,825; 5,661,016; and 5,545,806. In addition, companies such asAbgenix, Inc. (Freemont, Calif.), can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. This technology is described by Jespers etal. (1994) Bio/Technology 12:899-903).

An anti-adenylate kinase antibody (e.g., monoclonal antibody) can beused to isolate adenylate kinase proteins by standard techniques, suchas affinity chromatography or immunoprecipitation. An anti-adenylatekinase antibody can facilitate the purification of natural adenylatekinase protein from cells and of recombinantly produced adenylate kinaseprotein expressed in host cells. Moreover, an anti-adenylate kinaseantibody can be used to detect adenylate kinase protein (e.g., in acellular lysate or cell supernatant) in order to evaluate the abundanceand pattern of expression of the adenylate kinase protein.Anti-adenylate kinase antibodies can be used diagnostically to monitorprotein levels in tissue as part of a clinical testing procedure, e.g.,to, for example, determine the efficacy of a given treatment regimen.Detection can be facilitated by coupling the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S,or ³H.

Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). The conjugates of the invention canbe used for modifying a given biological response, the drug moiety isnot to be construed as limited to classical chemical therapeutic agents.For example, the drug moiety may be a protein or polypeptide possessinga desired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, .alpha.-interferon,.beta.-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al. (1985) “Monoclonal Antibodies forImmunotargeting of Drugs in Cancer Therapy,” in Monoclonal AntibodiesAnd Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.), pp.243-56); Hellstrom et al. (1987) “Antibodies for Drug Delivery,” inControlled Drug Delivery, ed. Robinson et al. (2d ed., Marcel Dekker,Inc.), pp. 623-53; Thorpe (1985) “Antibody Carriers of Cytotoxic Agentsin Cancer Therapy: A Review”, in Monoclonal Antibodies '84: BiologicalAnd Clinical Applications, ed. Pinchera et al., pp. 475-506; “Analysis,Results, and Future Prospective of the Therapeutic Use of RadiolabeledAntibody in Cancer Therapy,” in Monoclonal Antibodies For CancerDetection And Therapy, ed. Baldwin et al. (Academic Press, NY), pp.303-316; and Thorpe et al. (1982) Immunol. Rev. 62:119-58.Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding an adenylatekinase protein (or a portion thereof). “Vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked, such as a “plasmid”, a circular double-stranded DNA loopinto which additional DNA segments can be ligated, or a viral vector,where additional DNA segments can be ligated into the viral genome. Thevectors are useful for autonomous replication in a host cell or may beintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome (e.g.,nonepisomal mammalian vectors). Expression vectors are capable ofdirecting the expression of genes to which they are operably linked. Ingeneral, expression vectors of utility in recombinant DNA techniques areoften in the form of plasmids (vectors). However, the invention isintended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenoviruses,and adeno-associated viruses), that serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell. This means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, operably linked to the nucleicacid sequence to be expressed. “Operably linked” is intended to meanthat the nucleotide sequence of interest is linked to the regulatorysequence(s) in a manner that allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in ahost cell when the vector is introduced into the host cell). The term“regulatory sequence” is intended to include promoters, enhancers, andother expression control elements (e.g., polyadenylation signals). See,for example, Goeddel (1990) in Gene Expression Technology: Methods inEnzymology 185 (Academic Press, San Diego, Calif.). Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cell and those that direct expression ofthe nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., adenylate kinase proteins,mutant forms of adenylate kinase proteins, fusion proteins, etc.).

It is further recognized that the nucleic acid sequences of theinvention can be altered to contain codons, which are preferred, or nonpreferred, for a particular expression system. For example, the nucleicacid can be one in which at least one altered codon, and preferably atleast 10%, or 20% of the codons have been altered such that the sequenceis optimized for expression in E. coli, yeast, human, insect, or CHOcells. Methods for determining such codon usage are well known in theart.

The recombinant expression vectors of the invention can be designed forexpression of adenylate kinase protein in prokaryotic or eukaryotic hostcells. Expression of proteins in prokaryotes is most often carried outin E. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or nonfusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Typical fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly,Mass.), and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein. Examples of suitableinducible nonfusion E. coli expression vectors include pTrc (Amann etal. (1988) Gene 69:301-315) and pET 11d (Studier et al. (1990) in GeneExpression Technology: Methods in Enzymology 185 (Academic Press, SanDiego, Calif.), pp. 60-89). Strategies to maximize recombinant proteinexpression in E. coli can be found in Gottesman (1990) in GeneExpression Technology: Methods in Enzymology 185 (Academic Press, CA),pp. 119-128 and Wada et al. (1992) Nucleic Acids Res. 20:2111-2118.Target gene expression from the pTrc vector relies on host RNApolymerase transcription from a hybrid trp-lac fusion promoter.

Suitable eukaryotic host cells include insect cells (examples ofBaculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39)); yeast cells (examples of vectorsfor expression in yeast S. cereivisiae include pYepSec1 (Baldari et al.(1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2(Invitrogen Corporation, San Diego, Calif.), and pPicZ (InvitrogenCorporation, San Diego, Calif.)); or mammalian cells (mammalianexpression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC(Kaufman et al. (1987) EMBO J. 6:187:195)). Suitable mammalian cellsinclude Chinese hamster ovary cells (CHO) or COS cells. In mammaliancells, the expression vector's control functions are often provided byviral regulatory elements. For example, commonly used promoters arederived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus40. For other suitable expression systems for both prokaryotic andeukaryotic cells, see chapters 16 and 17 of Sambrook et al. (1989)Molecular cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.). See, Goeddel (1990) in GeneExpression Technology: Methods in Enzymology 185 (Academic Press, SanDiego, Calif.). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell but are stillincluded within the scope of the term as used herein. A “purifiedpreparation of cells”, as used herein, refers to, in the case of plantor animal cells, an in vitro preparation of cells and not an entireintact plant or animal. In the case of cultured cells or microbialcells, it consists of a preparation of at least 10% and more preferably50% of the subject cells.

In one embodiment, the expression vector is a recombinant mammalianexpression vector that comprises tissue-specific regulatory elementsthat direct expression of the nucleic acid preferentially in aparticular cell type. Suitable tissue-specific promoters include thealbumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev.1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv.Immunol. 43:235-275), in particular promoters of T cell receptors(Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins(Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell33:741-748), neuron-specific promoters (e.g., the neurofilamentpromoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science230:912-916), and mammary gland-specific promoters (e.g., milk wheypromoter; U.S. Pat. No. 4,873,316 and European Application PatentPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379), the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546), and the like.

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperably linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to adenylate kinase mRNA. Regulatory sequencesoperably linked to a nucleic acid cloned in the antisense orientationcan be chosen to direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen to direct constitutive,tissue-specific, or cell-type-specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid, or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub et al. (1986)Reviews—Trends in Genetics, Vol. 1(1).

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.) and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin, and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding an adenylate kinase protein or can be introducedon a separate vector. Cells stably transfected with the introducednucleic acid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) adenylate kinaseprotein. Accordingly, the invention further provides methods forproducing adenylate kinase protein using the host cells of theinvention. In one embodiment, the method comprises culturing the hostcell of the invention, into which a recombinant expression vectorencoding an adenylate kinase protein has been introduced, in a suitablemedium such that adenylate kinase protein is produced. In anotherembodiment, the method further comprises isolating adenylate kinaseprotein from the medium or the host cell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. In general, methods for producing transgenic animalsinclude introducing a nucleic acid sequence according to the presentinvention, the nucleic acid sequence: capable of expressing the receptorprotein in a transgenic animal, into a cell in culture or in vivo. Whenintroduced in vivo, the nucleic acid is introduced into an intactorganism such that one or more cell types and, accordingly, one or moretissue types, express the nucleic acid encoding the receptor protein.Alternatively, the nucleic acid can be introduced into virtually allcells in an organism by transfecting a cell in culture, such as anembryonic stem cell, as described herein for the production oftransgenic animals, and this cell can be used to produce an entiretransgenic organism. As described, in a further embodiment, the hostcell can be a fertilized oocyte. Such cells are then allowed to developin a female foster animal to produce the transgenic organism.

For example, in one embodiment, a host cell of the invention is afertilized oocyte or an embryonic stem cell into which adenylatekinase-coding sequences have been introduced. Such host cells can thenbe used to create nonhuman transgenic animals in which exogenousadenylate kinase sequences have been introduced into their genome orhomologous recombinant animals in which endogenous adenylate kinasesequences have been altered. Such animals are useful for studying thefunction and/or activity of adenylate kinase genes and proteins and foridentifying and/or evaluating modulators of adenylate kinase activity.As used herein, a “transgenic animal” is a nonhuman animal, preferably amammal, more preferably a rodent such as a rat or mouse, in which one ormore of the cells of the animal includes a transgene. Other examples oftransgenic animals include nonhuman primates, sheep, dogs, cows, goats,chickens, amphibians, etc. A transgene is exogenous DNA that isintegrated into the genome of a cell from which a transgenic animaldevelops and which remains in the genome of the mature animal, therebydirecting the expression of an encoded gene product in one or more celltypes or tissues of the transgenic animal. As used herein, a “homologousrecombinant animal” is a nonhuman animal, preferably a mammal, morepreferably a mouse, in which an endogenous adenylate kinase gene hasbeen altered by homologous recombination between the endogenous gene andan exogenous DNA molecule introduced into a cell of the animal, e.g., anembryonic cell of the animal, prior to development of the animal.

A transgenic animal of the invention can be created by introducingadenylate kinase-encoding nucleic acid into the male pronuclei of afertilized oocyte, e.g., by microinjection, retroviral infection, andallowing the oocyte to develop in a pseudopregnant female foster animal.The adenylate kinase cDNA sequence can be introduced as a transgene intothe genome of a nonhuman animal. Alternatively, a homolog of the mouseadenylate kinase gene can be isolated based on hybridization and used asa transgene. Intronic sequences and polyadenylation signals can also beincluded in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operablylinked to the adenylate kinase transgene to direct expression ofadenylate kinase protein to particular cells. Methods for generatingtransgenic animals via embryo manipulation and microinjection,particularly animals such as mice, have become conventional in the artand are described, for example, in U.S. Pat. Nos. 4,736,866, 4,870,009,and 4,873,191 and in Hogan (1986) Manipulating the Mouse Embryo (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similarmethods are used for production of other transgenic animals. Atransgenic founder animal can be identified based upon the presence ofthe adenylate kinase transgene in its genome and/or expression ofadenylate kinase mRNA in tissues or cells of the animals. A transgenicfounder animal can then be used to breed additional animals carrying thetransgene. Moreover, transgenic animals carrying a transgene encodingadenylate kinase gene can further be bred to other transgenic animalscarrying other transgenes.

To create a homologous recombinant animal, one prepares a vectorcontaining at least a portion of an adenylate kinase gene or a homologof the gene into which a deletion, addition, or substitution has beenintroduced to thereby alter, e.g., functionally disrupt, the adenylatekinase gene. In a preferred embodiment, the vector is designed suchthat, upon homologous recombination, the endogenous adenylate kinasegene is functionally disrupted (i.e., no longer encodes a functionalprotein; also referred to as a “knock out” vector). Alternatively, thevector can be designed such that, upon homologous recombination, theendogenous adenylate kinase gene is mutated or otherwise altered butstill encodes functional protein (e.g., the upstream regulatory regioncan be altered to thereby alter the expression of the endogenousadenylate kinase protein). In the homologous recombination vector, thealtered portion of the adenylate kinase gene is flanked at its 5′ and 3′ends by additional nucleic acid of the adenylate kinase gene to allowfor homologous recombination to occur between the exogenous adenylatekinase gene carried by the vector and an endogenous adenylate kinasegene in an embryonic stem cell. The additional flanking adenylate kinasenucleic acid is of sufficient length for successful homologousrecombination with the endogenous gene. Typically, several kilobases offlanking DNA (both at the 5′ and 3′ ends) are included in the vector(see, e.g., Thomas and Capecchi (1987) Cell 51:503 for a description ofhomologous recombination vectors). The vector is introduced into anembryonic stem cell line (e.g., by electroporation), and cells in whichthe introduced adenylate kinase gene has homologously recombined withthe endogenous adenylate kinase gene are selected (see, e.g., Li et al.(1992) Cell 69:915). The selected cells are then injected into ablastocyst of an animal (e.g., a mouse) to form aggregation chimeras(see, e.g., Bradley (1987) in Teratocarcinomas and Embryonic Stem Cells:A Practical Approach, ed. Robertson (IRL, Oxford), pp. 113-152). Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term. Progeny harboringthe homologously recombined DNA in their germ cells can be used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA by germline transmission of the transgene. Methods forconstructing homologous recombination vectors and homologous recombinantanimals are described further in Bradley (1991) Current Opinion inBio/Technology 2:823-829 and in PCT Publication Nos. WO 90/11354, WO91/01140, WO 92/0968, and WO 93/04169.

In another embodiment, transgenic nonhuman animals containing selectedsystems that allow for regulated expression of the transgene can beproduced. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355). If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the nonhuman transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and PCT Publication Nos. WO 97/07668 and WO 97/07669.

IV. Pharmaceutical Compositions

The adenylate kinase nucleic acid molecules, adenylate kinase proteins,and anti-adenylate kinase antibodies (also referred to herein as “activecompounds”) of the invention can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise the nucleic acid molecule, protein, or antibody and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The compositions of the invention are useful to treat any of thedisorders discussed herein. The compositions are provided intherapeutically effective amounts. By “therapeutically effectiveamounts” is intended an amount sufficient to modulate the desiredresponse. As defined herein, a therapeutically effective amount ofprotein or polypeptide (i.e., an effective dosage) ranges from about0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg bodyweight, more preferably about 0.1 to 20 mg/kg body weight, and even morepreferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7mg/kg, or 5 to 6 mg/kg body weight.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of a protein, polypeptide, or antibody can include asingle treatment or, preferably, can include a series of treatments. Ina preferred example, a subject is treated with antibody, protein, orpolypeptide in the range of between about 0.1 to 20 mg/kg body weight,one time per week for between about 1 to 10 weeks, preferably between 2to 8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody, protein, or polypeptide used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays as described herein.

The present invention encompasses agents that modulate expression oractivity. An agent may, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e,. including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds.

It is understood that appropriate doses of small molecule agents dependsupon a number of factors within the ken of the ordinarily skilledphysician, veterinarian, or researcher. The dose(s) of the smallmolecule will vary, for example, depending upon the identity, size, andcondition of the subject or sample being treated, further depending uponthe route by which the composition is to be administered, if applicable,and the effect which the practitioner desires the small molecule to haveupon the nucleic acid or polypeptide of the invention. Exemplary dosesinclude milligram or microgram amounts of the small molecule perkilogram of subject or sample weight (e.g., about 1 microgram perkilogram to about 500 milligrams per kilogram, about 100 micrograms perkilogram to about 5 milligrams per kilogram, or about 1 microgram perkilogram to about 50 micrograms per kilogram. It is furthermoreunderstood that appropriate doses of a small molecule depend upon thepotency of the small molecule with respect to the expression or activityto be modulated. Such appropriate doses may be determined using theassays described herein. When one or more of these small molecules is tobe administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes, or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF; Parsippany, N.J.), or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion, and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., an adenylate kinase protein or anti-adenylate kinaseantibody) in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth, or gelatin; an excipientsuch as starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. For administrationby inhalation, the compounds are delivered in the form of an aerosolspray from a pressurized container or dispenser that contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art. Thecompounds can also be prepared in the form of suppositories (e.g., withconventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated with each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. Depending on thetype and severity of the disease, about 1 μg/kg to about 15 mg/kg (e.g.,0.1 to 20 mg/kg) of antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to about 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays. Anexemplary dosing regimen is disclosed in WO 94/04188. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470), or by stereotactic injection(see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of a protein, polypeptide, or antibody can include asingle treatment or, preferably, can include a series of treatments. Ina preferred example, a subject is treated with antibody, protein, orpolypeptide in the range of between about 0.1 to 20 mg/kg body weight,one time per week for between about 1 to 10 weeks, preferably between 2to 8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody, protein, or polypeptide used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays as described herein.

The present invention encompasses agents which modulate expression oractivity. An agent may, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e., including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds.

It is understood that appropriate doses of small molecule agents dependsupon a number of factors within the ken of the ordinarily skilledphysician, veterinarian, or researcher. The dose(s) of the smallmolecule will vary, for example, depending upon the identity, size, andcondition of the subject or sample being treated, further depending uponthe route by which the composition is to be administered, if applicable,and the effect which the practitioner desires the small molecule to haveupon the nucleic acid or polypeptide of the invention. Exemplary dosesinclude milligram or microgram amounts of the small molecule perkilogram of subject or sample weight (e.g., about 1 microgram perkilogram to about 500 milligrams per kilogram, about 100 micrograms perkilogram to about 5 milligrams per kilogram, or about 1 microgram perkilogram to about 50 micrograms per kilogram. It is furthermoreunderstood that appropriate doses of a small molecule depend upon thepotency of the small molecule with respect to the expression or activityto be modulated. Such appropriate doses may be determined using theassays described herein. When one or more of these small molecules is tobe administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

Computer Readable Means

The nucleotide or amino acid sequences of the invention are alsoprovided in a variety of mediums to facilitate use thereof. As usedherein, “provided” refers to a manufacture, other than an isolatednucleic acid or amino acid molecule, which contains a nucleotide oramino acid sequence of the present invention. Such a manufactureprovides the nucleotide or amino acid sequences, or a subset thereof(e.g., a subset of open reading frames (ORFs)) in a form which allows askilled artisan to examine the manufacture using means not directlyapplicable to examining the nucleotide or amino acid sequences, or asubset thereof, as they exists in nature or in purified form.

In one application of this embodiment, a nucleotide or amino acidsequence of the present invention can be recorded on computer readablemedia. As used herein, “computer readable media” refers to any mediumthat can be read and accessed directly by a computer. Such mediainclude, but are not limited to: magnetic storage media, such as floppydiscs, hard disc storage medium, and magnetic tape; optical storagemedia such as CD-ROM; electrical storage media such as RAM and ROM; andhybrids of these categories such as magnetic/optical storage media. Theskilled artisan will readily appreciate how any of the presently knowncomputer readable mediums can be used to create a manufacture comprisingcomputer readable medium having recorded thereon a nucleotide or aminoacid sequence of the present invention.

As used herein, “recorded” refers to a process for storing informationon computer readable medium. The skilled artisan can readily adopt anyof the presently known methods for recording information on computerreadable medium to generate manufactures comprising the nucleotide oramino acid sequence information of the present invention.

A variety of data storage structures are available to a skilled artisanfor creating a computer readable medium having recorded thereon anucleotide or amino acid sequence of the present invention. The choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats can be used to store the nucleotidesequence information of the present invention on computer readablemedium. The sequence information can be represented in a word processingtext file, formatted in commercially-available software such asWordPerfect and MicroSoft Word, or represented in the form of an ASCIIfile, stored in a database application, such as DB2, Sybase, Oracle, orthe like. The skilled artisan can readily adapt any number ofdataprocessor structuring formats (e.g., text file or database) in orderto obtain computer readable medium having recorded thereon thenucleotide sequence information of the present invention.

By providing the nucleotide or amino acid sequences of the invention incomputer readable form, the skilled artisan can routinely access thesequence information for a variety of purposes. For example, one skilledin the art can use the nucleotide or amino acid sequences of theinvention in computer readable form to compare a target sequence ortarget structural motif with the sequence information stored within thedata storage means. Search means are used to identify fragments orregions of the sequences of the invention which match a particulartarget sequence or target motif.

As used herein, a “target sequence” can be any DNA or amino acidsequence of six or more nucleotides or two or more amino acids. Askilled artisan can readily recognize that the longer a target sequenceis, the less likely a target sequence will be present as a randomoccurrence in the database. The most preferred sequence length of atarget sequence is from about 10 to 100 amino acids or from about 30 to300 nucleotide residues. However, it is well recognized thatcommercially important fragments, such as sequence fragments involved ingene expression and protein processing, may be of shorter length.

As used herein, “a target structural motif,” or “target motif,” refersto any rationally selected sequence or combination of sequences in whichthe sequence(s) are chosen based on a three-dimensional configurationwhich is formed upon the folding of the target motif. There are avariety of target motifs known in the art. Protein target motifsinclude, but are not limited to, enzyme active sites and signalsequences. Nucleic acid target motifs include, but are not limited to,promoter sequences, hairpin structures and inducible expression elements(protein binding sequences).

Computer software is publicly available which allows a skilled artisanto access sequence information provided in a computer readable mediumfor analysis and comparison to other sequences. A variety of knownalgorithms are disclosed publicly and a variety of commerciallyavailable software for conducting search means are and can be used inthe computer-based systems of the present invention. Examples of suchsoftware includes, but is not limited to, MacPattern (EMBL), BLASTN andBLASTX (NCBIA).

For example, software which implements the BLAST (Altschul et al. (1990)J. Mol. Biol. 215:403-410) and BLAZE (Brutlag et al. (1993) Comp. Chem.17:203-207) search algorithms on a Sybase system can be used to identifyopen reading frames (ORFs) of the sequences of the invention whichcontain homology to ORFs or proteins from other libraries. Such ORFs areprotein encoding fragments and are useful in producing commerciallyimportant proteins such as enzymes used in various reactions and in theproduction of commercially useful metabolites.

V. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologs, and antibodiesdescribed herein can be used in one or more of the following methods:(a) screening assays; (b) detection assays (e.g., chromosomal mapping,tissue typing, forensic biology); (c) predictive medicine (e.g.,diagnostic assays, prognostic assays, monitoring clinical trials, andpharmacogenomics); and (d) methods of treatment (e.g., therapeutic andprophylactic). The uses and methods of the invention are particularlyrelevant in tissues and cells in which the adenylate kinase is expressedand especially where expression differs from that in a normal tissue orcell. The uses and methods are also particularly relevant in disordersinvolving such tissues and cells. Accordingly, the uses and methods areparticularly relevant for disorders involving expression of theadenylate kinase of the invention. The isolated nucleic acid moleculesof the invention can be used to express adenylate kinase protein (e.g.,via a recombinant expression vector in a host cell in gene therapyapplications), to detect adenylate kinase mRNA (e.g., in a biologicalsample) or a genetic lesion in an adenylate kinase gene, and to modulateadenylate kinase activity. In addition, the adenylate kinase proteinscan be used to screen drugs or compounds that modulate the immuneresponse as well as to treat disorders characterized by insufficient orexcessive production of adenylate kinase protein or production ofadenylate kinase protein forms that have decreased or aberrant activitycompared to adenylate kinase wild type protein. In addition, theanti-adenylate kinase antibodies of the invention can be used to detectand isolate adenylate kinase proteins and modulate adenylate kinaseactivity.

A. Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules, or otherdrugs) that bind to adenylate kinase proteins or have a stimulatory orinhibitory effect on, for example, adenylate kinase expression oradenylate kinase activity.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including biological libraries, spatially addressable parallelsolid phase or solution phase libraries, synthetic library methodsrequiring deconvolution, the “one-bead one-compound” library method, andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, nonpeptide oligomer, orsmall molecule libraries of compounds (Lam (1997) Anticancer Drug Des.12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA.91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Bio/Techniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Pat.No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA89:1865-1869), or phage (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. USA 87:6378-6382; and Felici (1991) J. Mol. Biol.222:301-310).

Determining the ability of the test compound to bind to the adenylatekinase protein can be accomplished, for example, by coupling the testcompound with a radioisotope or enzymatic label such that binding of thetest compound to the adenylate kinase protein or biologically activeportion thereof can be determined by detecting the labeled compound in acomplex. For example, test compounds can be labeled with ¹²⁵I, ³⁵S, ¹⁴C,or ³H, either directly or indirectly, and the radioisotope detected bydirect counting of radioemmission or by scintillation counting.Alternatively, test compounds can be enzymatically labeled with, forexample, horseradish peroxidase, alkaline phosphatase, or luciferase,and the enzymatic label detected by determination of conversion of anappropriate substrate to product.

In a similar manner, one may determine the ability of the adenylatekinase protein to bind to or interact with an adenylate kinase targetmolecule. By “target molecule” is intended a molecule with which anadenylate kinase protein binds or interacts in nature. In a preferredembodiment, the ability of the adenylate kinase protein to bind to orinteract with an adenylate kinase target molecule can be determined bymonitoring the activity of the target molecule. For example, theactivity of the target molecule can be monitored by detecting inductionof a cellular second messenger of the target (e.g., intracellular Ca²⁺,diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity ofthe target on an appropriate substrate, detecting the induction of areporter gene (e.g., an adenylate kinase-responsive regulatory elementoperably linked to a nucleic acid encoding a detectable marker, e.g.luciferase), or detecting a cellular response, for example, cellulardifferentiation or cell proliferation.

In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting an adenylate kinase protein orbiologically active portion thereof with a test compound and determiningthe ability of the test compound to bind to the adenylate kinase proteinor biologically active portion thereof. Binding of the test compound tothe adenylate kinase protein can be determined either directly orindirectly as described above. In a preferred embodiment, the assayincludes contacting the adenylate kinase protein or biologically activeportion thereof with a known compound that binds adenylate kinaseprotein to form an assay mixture, contacting the assay mixture with atest compound, and determining the ability of the test compound topreferentially bind to adenylate kinase protein or biologically activeportion thereof as compared to the known compound.

In another embodiment, an assay is a cell-free assay comprisingcontacting adenylate kinase protein or biologically active portionthereof with a test compound and determining the ability of the testcompound to modulate (e.g., stimulate or inhibit) the activity of theadenylate kinase protein or biologically active portion thereof.Determining the ability of the test compound to modulate the activity ofan adenylate kinase protein can be accomplished, for example, bydetermining the ability of the adenylate kinase protein to bind to anadenylate kinase target molecule as described above for determiningdirect binding. In an alternative embodiment, determining the ability ofthe test compound to modulate the activity of an adenylate kinaseprotein can be accomplished by determining the ability of the adenylatekinase protein to further modulate an adenylate kinase target molecule.For example, the catalytic/enzymatic activity of the target molecule onan appropriate substrate can be determined as previously described.

In yet another embodiment, the cell-free assay comprises contacting theadenylate kinase protein or biologically active portion thereof with aknown compound that binds an adenylate kinase protein to form an assaymixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to preferentially bind toor modulate the activity of an adenylate kinase target molecule.

In the above-mentioned assays, it may be desirable to immobilize eitheran adenylate kinase protein or its target molecule to facilitateseparation of complexed from uncomplexed forms of one or both of theproteins, as well as to accommodate automation of the assay. In oneembodiment, a fusion protein can be provided that adds a domain thatallows one or both of the proteins to be bound to a matrix. For example,glutathione-S-transferase/adenylate kinase fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione-derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the nonadsorbed targetprotein or adenylate kinase protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components andcomplex formation is measured either directly or indirectly, forexample, as described above. Alternatively, the complexes can bedissociated from the matrix, and the level of adenylate kinase bindingor activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either adenylatekinase protein or its target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated adenylate kinasemolecules or target molecules can be prepared frombibtin-NHS(N-hydroxy-succinimide) using techniques well known in the art(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96-well plates (PierceChemicals). Alternatively, antibodies reactive with an adenylate kinaseprotein or target molecules but which do not interfere with binding ofthe adenylate kinase protein to its target molecule can be derivatizedto the wells of the plate, and unbound target or adenylate kinaseprotein trapped in the wells by antibody conjugation. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the adenylate kinase protein or targetmolecule, as well as enzyme-linked assays that rely on detecting anenzymatic activity associated with the adenylate kinase protein ortarget molecule.

In another embodiment, modulators of adenylate kinase expression areidentified in a method in which a cell is contacted with a candidatecompound and the expression of adenylate kinase mRNA or protein in thecell is determined relative to expression of adenylate kinase mRNA orprotein in a cell in the absence of the candidate compound. Whenexpression is greater (statistically significantly greater) in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of adenylate kinase mRNA orprotein expression. Alternatively, when expression is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of adenylate kinase mRNA or protein expression. The level ofadenylate kinase mRNA or protein expression in the cells can bedetermined by methods described herein for detecting adenylate kinasemRNA or protein.

In yet another aspect of the invention, the adenylate kinase proteinscan be used as “bait proteins” in a two-hybrid assay or three-hybridassay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993). Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Bio/Techniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and PCT Publication No. WO 94/10300), to identify otherproteins, which bind to or interact with adenylate kinase protein(“adenylate kinase-binding proteins” or “adenylate kinase-bp”) andmodulate adenylate kinase activity. Such adenylate kinase-bindingproteins are also likely to be involved in the propagation of signals bythe adenylate kinase proteins as, for example, upstream or downstreamelements of the adenylate kinase pathway.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein. Accordingly the invention is directed to agents thatmodulate the level or activity of the polypeptide or nucleic acid of theinvention, the agents being identified by screening cells, tissues, cellextracts, or tissue extracts with the agents. Agents that alter thelevel or activity can then be tested further for clinical diagnostic ortherapeutic use. Any method of screening that allows expression to bemeasured, such as those disclosed herein, are relevant to produce theidentification of these agents. Thus, the invention is directed toagents identified by the screening processes involving measuring ordetecting expression (level or activity) of the polypeptides or nucleicacids of the invention. It is understood that agents affecting theability of the protein or nucleic acid to interact with a cellularcomponent, as in competition binding, would be construed as affectingexpression. Accordingly, screening processes also include assays foragents that themselves bind to the protein or nucleic acid of theinvention, such as those disclosed herein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(1) map their respective genes on a chromosome; (2) identify anindividual from a minute biological sample (tissue typing); and (3) aidin forensic identification of a biological sample. These applicationsare described in the subsections below.

1. Chromosome Mapping

The isolated complete or partial adenylate kinase gene sequences of theinvention can be used to map their respective adenylate kinase genes ona chromosome, thereby facilitating the location of gene regionsassociated with genetic disease. Computer analysis of adenylate kinasesequences can be used to rapidly select PCR primers (preferably 15-25 bpin length) that do not span more than one exon in the genomic DNA,thereby simplifying the amplification process. These primers can then beused for PCR screening of somatic cell hybrids containing individualhuman chromosomes. Only those hybrids containing the human genecorresponding to the adenylate kinase sequences will yield an amplifiedfragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow (because they lack a particular enzyme), but in whichhuman cells can, the one human chromosome that contains the geneencoding the needed enzyme will be retained. By using various media,panels of hybrid cell lines can be established. Each cell line in apanel contains either a single human chromosome or a small number ofhuman chromosomes, and a full set of mouse chromosomes, allowing easymapping of individual genes to specific human chromosomes (D'Eustachioet al. (1983) Science 220:919-924). Somatic cell hybrids containing onlyfragments of human chromosomes can also be produced by using humanchromosomes with translocations and deletions.

Other mapping strategies that can similarly be used to map an adenylatekinase sequence to its chromosome include in situ hybridization(described in Fan et al. (1990) Proc. Natl. Acad. Sci. USA 87:6223-27),pre-screening with labeled flow-sorted chromosomes, and pre-selection byhybridization to chromosome specific cDNA libraries. Furthermore,fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. For a review of this technique, seeVerma eta a. (1988) Human Chromosomes: A Manual of Basic Techniques(Pergamon Press, NY). The FISH technique can be used with a DNA sequenceas short as 500 or 600 bases. However, clones larger than 1,000 baseshave a higher likelihood of binding to a unique chromosomal locationwith sufficient signal intensity for simple detection. Preferably 1,000bases, and more preferably 2,000 bases will suffice to get good resultsin a reasonable amount of time.

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship betweengenes and disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, e.g., Egeland et al. (1987) Nature325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the adenylate kinase genecan be determined. If a mutation is observed in some or all of theaffected individuals but not in any unaffected individuals, then themutation is likely to be the causative agent of the particular disease.Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes such asdeletions or translocations that are visible from chromosome spreads ordetectable using PCR based on that DNA sequence. Ultimately, completesequencing of genes from several individuals can be performed to confirmthe presence of a mutation and to distinguish mutations frompolymorphisms.

2. Tissue Typing

The adenylate kinase sequences of the present invention can also be usedto identify individuals from minute biological samples. The UnitedStates military, for example, is considering the use of restrictionfragment length polymorphism (RFLP) for identification of its personnel.In this technique, an individual's genomic DNA is digested with one ormore restriction enzymes and probed on a Southern blot to yield uniquebands for identification. The sequences of the present invention areuseful as additional DNA markers for RFLP (described in U.S. Pat. No.5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique for determining the actual base-by-baseDNA sequence of selected portions of an individual's genome. Thus, theadenylate kinase sequences of the invention can be used to prepare twoPCR primers from the 5′ and 3′ ends of the sequences. These primers canthen be used to amplify an individual's DNA and subsequently sequenceit.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The adenylate kinase sequences of the invention uniquelyrepresent portions of the human genome. Allelic variation occurs to somedegree in the coding regions of these sequences, and to a greater degreein the noncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency of about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. The noncoding sequences of SEQ ID NO:21 cancomfortably provide positive individual identification with a panel ofperhaps 10 to 1,000 primers that each yield a noncoding amplifiedsequence of 100 bases. If a predicted coding sequence, such as that inSEQ ID NO:21, is used, a more appropriate number of primers for positiveindividual identification would be 500 to 2,000.

3. Use of Partial Adenylate Kinase Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. In this manner, PCR technology can be used to amplify DNAsequences taken from very small biological samples such as tissues,e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen foundat a crime scene. The amplified sequence can then be compared to astandard, thereby allowing identification of the origin of thebiological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” that is unique to a particular individual. Asmentioned above, actual base sequence information can be used foridentification as an accurate alternative to patterns formed byrestriction enzyme generated fragments. Sequences targeted to noncodingregions of SEQ ID NO:21 are particularly appropriate for this use asgreater numbers of polymorphisms occur in the noncoding regions, makingit easier to differentiate individuals using this technique. Examples ofpolynucleotide reagents include the adenylate kinase sequences orportions thereof, e.g., fragments derived from the noncoding regions ofSEQ ID NO:21 having a length of at least 20 or 30 bases.

The adenylate kinase sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes thatcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue. This can be very useful in cases where aforensic pathologist is presented with a tissue of unknown origin.Panels of such adenylate kinase probes, can be used to identify tissueby species and/or by organ type.

In a similar fashion, these reagents, e.g., adenylate kinase primers orprobes can be used to screen tissue culture for contamination (i.e.,screen for the presence of a mixture of different types of cells in aculture).

C. Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trails are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. These applications aredescribed in the subsections below.

1. Diagnostic Assays

One aspect of the present invention relates to diagnostic assays fordetecting adenylate kinase protein and/or nucleic acid expression aswell as adenylate kinase activity, in the context of a biologicalsample. An exemplary method for detecting the presence or absence ofadenylate kinase proteins in a biological sample involves obtaining abiological sample from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting adenylate kinaseprotein or nucleic acid (e.g., mRNA, genomic DNA) that encodes adenylatekinase protein such that the presence of adenylate kinase protein isdetected in the biological sample. Results obtained with a biologicalsample from the test subject may be compared to results obtained with abiological sample from a control subject.

“Misexpression or aberrant expression”, as used herein, refers to anon-wild type pattern of gene expression, at the RNA or protein level.It includes: expression at non-wild type levels; i.e., over or underexpression; a pattern of expression that differs from wild type in termsof the time or stage at which the gene is expressed, e.g., increased ordecreased expression (as compared with wild type) at a predetermineddevelopmental period or stage; a pattern of expression that differs fromwild type in terms of decreased expression (as compared with wild type)in a predetermined cell type or tissue type; a pattern of expressionthat differs from wild type in terms of the splicing size, amino acidsequence, post-transitional modification, or biological activity of theexpressed polypeptide; a pattern of expression that differs from wildtype in terms of the effect of an environmental stimulus orextracellular stimulus on expression of the gene, e.g., a pattern ofincreased or decreased expression (as compared with wild type) in thepresence of an increase or decrease in the strength of the stimulus.

A preferred agent for detecting adenylate kinase mRNA or genomic DNA isa labeled nucleic acid probe capable of hybridizing to adenylate kinasemRNA or genomic DNA. The nucleic acid probe can be, for example, afull-length adenylate kinase nucleic acid, such as the nucleic acid ofSEQ ID NO:21, or a portion thereof, such as a nucleic acid molecule ofat least about 15, 30, 50, 100, 250, or 500 nucleotides in length andsufficient to specifically hybridize under stringent conditions toadenylate kinase mRNA or genomic DNA. Other suitable probes for use inthe diagnostic assays of the invention are described herein.

A preferred agent for detecting adenylate kinase protein is an antibodycapable of binding to adenylate kinase protein, preferably an antibodywith a detectable label. Antibodies can be polyclonal, or morepreferably, monoclonal. An intact antibody, or a fragment thereof (e.g.,Fab or F(abN)₂) can be used. The term “labeled”, with regard to theprobe or antibody, is intended to encompass direct labeling of the probeor antibody by coupling (i.e., physically linking) a detectablesubstance to the probe or antibody, as well as indirect labeling of theprobe or antibody by reactivity with another reagent that is directlylabeled. Examples of indirect labeling include detection of a primaryantibody using a fluorescently labeled secondary antibody andend-labeling of a DNA probe with biotin such that it can be detectedwith fluorescently labeled streptavidin.

The term “biological sample” is intended to include tissues, cells, andbiological fluids isolated from a subject, as well as tissues, cells,and fluids present within a subject. That is, the detection method ofthe invention can be used to detect adenylate kinase mRNA, protein, orgenomic DNA in a biological sample in vitro as well as in vivo. Forexample, in vitro techniques for detection of adenylate kinase mRNAinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of adenylate kinase protein include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, and immunofluorescence. In vitro techniques fordetection of adenylate kinase genomic DNA include Southernhybridizations. Furthermore, in vivo techniques for detection ofadenylate kinase protein include introducing into a subject a labeledanti-adenylate kinase antibody. For example, the antibody can be labeledwith a radioactive marker whose presence and location in a subject canbe detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a peripheral blood leukocytesample isolated by conventional means from a subject.

The invention also encompasses kits for detecting the presence ofadenylate kinase proteins in a biological sample (a test sample). Suchkits can be used to determine if a subject is suffering from or is atincreased risk of developing a disorder associated with aberrantexpression of adenylate kinase protein (e.g., an immunologicaldisorder). For example, the kit can comprise a labeled compound or agentcapable of detecting adenylate kinase protein or mRNA in a biologicalsample and means for determining the amount of an adenylate kinaseprotein in the sample (e.g., an anti-adenylate kinase antibody or anoligonucleotide probe that binds to DNA encoding an adenylate kinaseprotein, e.g., SEQ ID NO:21). Kits can also include instructions forobserving that the tested subject is suffering from or is at risk ofdeveloping a disorder associated with aberrant expression of adenylatekinase sequences if the amount of adenylate kinase protein or mRNA isabove or below a normal level.

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) that binds to adenylatekinase protein; and, optionally, (2) a second, different antibody thatbinds to adenylate kinase protein or the first antibody and isconjugated to a detectable agent. For oligonucleotide-based kits, thekit can comprise, for example: (1) an oligonucleotide, e.g., adetectably labeled oligonucleotide, that hybridizes to an adenylatekinase nucleic acid sequence or (2) a pair of primers useful foramplifying an adenylate kinase nucleic acid molecule.

The kit can also comprise, e.g., a buffering agent, a preservative, or aprotein stabilizing agent. The kit can also comprise componentsnecessary for detecting the detectable agent (e.g., an enzyme or asubstrate). The kit can also contain a control sample or a series ofcontrol samples that can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container, and all of the various containers are within asingle package along with instructions for observing whether the testedsubject is suffering from or is at risk of developing a disorderassociated with aberrant expression of adenylate kinase proteins.

2. Prognostic Assays

The methods described herein can furthermore be utilized as diagnosticor prognostic assays to identify subjects having or at risk ofdeveloping a disease or disorder associated with adenylate kinaseprotein, adenylate kinase nucleic acid expression, or adenylate kinaseactivity. Prognostic assays can be used for prognostic or predictivepurposes to thereby prophylactically treat an individual prior to theonset of a disorder characterized by or associated with adenylate kinaseprotein, adenylate kinase nucleic acid expression, or adenylate kinaseactivity.

Thus, the present invention provides a method in which a test sample isobtained from a subject, and adenylate kinase protein or nucleic acid(e.g., mRNA, genomic DNA) is detected, wherein the presence of adenylatekinase protein or nucleic acid is diagnostic for a subject having or atrisk of developing a disease or disorder associated with aberrantadenylate kinase expression or activity. As used herein, a “test sample”refers to a biological sample obtained from a subject of interest. Forexample, a test sample can be a biological fluid (e.g., serum), cellsample, or tissue.

Furthermore, using the prognostic assays described herein, the presentinvention provides methods for determining whether a subject can beadministered a specific agent (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, nucleic acid, small molecule, or otherdrug candidate) or class of agents (e.g., agents of a type that decreaseadenylate kinase activity) to effectively treat a disease or disorderassociated with aberrant adenylate kinase expression or activity. Inthis manner, a test sample is obtained and adenylate kinase protein ornucleic acid is detected. The presence of adenylate kinase protein ornucleic acid is diagnostic for a subject that can be administered theagent to treat a disorder associated with aberrant adenylate kinaseexpression or activity.

The methods of the invention can also be used to detect genetic lesionsor mutations in an adenylate kinase gene, thereby determining if asubject with the lesioned gene is at risk for a disorder characterizedby aberrant cell proliferation and/or differentiation. In preferredembodiments, the methods include detecting, in a sample of cells fromthe subject, the presence or absence of a genetic lesion or mutationcharacterized by at least one of an alteration affecting the integrityof a gene encoding an adenylate kinase-protein, or the misexpression ofthe adenylate kinase gene. For example, such genetic lesions ormutations can be detected by ascertaining the existence of at least oneof: (1) a deletion of one or more nucleotides from an adenylate kinasegene; (2) an addition of one or more nucleotides to an adenylate kinasegene; (3) a substitution of one or more nucleotides of an adenylatekinase gene; (4) a chromosomal rearrangement of an adenylate kinasegene; (5) an alteration in the level of a messenger RNA transcript of anadenylate kinase gene; (6) an aberrant modification of an adenylatekinase gene, such as of the methylation pattern of the genomic DNA; (7)the presence of a non-wild-type splicing pattern of a messenger RNAtranscript of an adenylate kinase gene; (8) a non-wild-type level of anadenylate kinase-protein; (9) an allelic loss of an adenylate kinasegene; and (10) an inappropriate post-translational modification of anadenylate kinase-protein. As described herein, there are a large numberof assay techniques known in the art that can be used for detectinglesions in an adenylate kinase gene. Any cell type or tissue, preferablyperipheral blood leukocytes, in which adenylate kinase proteins areexpressed may be utilized in the prognostic assays described herein.

In certain embodiments, detection of the lesion involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the adenylatekinase gene (see, e.g., Abravaya et al. (1995) Nucleic Acids Res.23:675-682). It is anticipated that PCR and/or LCR may be desirable touse as a preliminary amplification step in conjunction with any of thetechniques used for detecting mutations described herein.

Alternative amplification methods include self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in an adenylate kinase gene froma sample cell can be identified by alterations in restriction enzymecleavage patterns of isolated test sample and control DNA digested withone or more restriction endonucleases. Moreover, the use of sequencespecific ribozymes (see, e.g., U.S. Pat. No. 5,498,531) can be used toscore for the presence of specific mutations by development or loss of aribozyme cleavage site.

In other embodiments, genetic mutations in an adenylate kinase moleculecan be identified by hybridizing a sample and control nucleic acids,e.g., DNA or RNA, to high density arrays containing hundreds orthousands of oligonucleotides probes (Cronin et al. (1996) HumanMutation 7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759). Inyet another embodiment, any of a variety of sequencing reactions knownin the art can be used to directly sequence the adenylate kinase geneand detect mutations by comparing the sequence of the sample adenylatekinase gene with the corresponding wild-type (control) sequence.Examples of sequencing reactions include those based on techniquesdeveloped by Maxim and Gilbert ((1977) Proc. Natl. Acad. Sci. USA74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It isalso contemplated that any of a variety of automated sequencingprocedures can be utilized when performing the diagnostic assays ((1995)Bio/Techniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT Publication No. WO 94/16101; Cohen et al. (1996) Adv.Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem.Biotechnol. 38:147-159).

Other methods for detecting mutations in the adenylate kinase geneinclude methods in which protection from cleavage agents is used todetect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers etal. (1985) Science 230:1242). See, also Cotton et al. (1988) Proc. Natl.Acad. Sci. USA 85:4397; Saleeba et al. (1992) Methods Enzymol.217:286-295. In a preferred embodiment, the control DNA or RNA can belabeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more “DNA mismatch repair” enzymes that recognize mismatched basepairs in double-stranded DNA in defined systems for detecting andmapping point mutations in adenylate kinase cDNAs obtained from samplesof cells. See, e.g., Hsu et al. (1994) Carcinogenesis 15:1657-1662.According to an exemplary embodiment, a probe based on an adenylatekinase sequence, e.g., a wild-type adenylate kinase sequence, ishybridized to a cDNA or other DNA product from a test cell(s). Theduplex is treated with a DNA mismatch repair enzyme, and the cleavageproducts, if any, can be detected from electrophoresis protocols or thelike. See, e.g., U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in adenylate kinase genes. For example,single-strand conformation polymorphism (SSCP) may be used to detectdifferences in electrophoretic mobility between mutant and wild-typenucleic acids (Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766;see also Cotton (1993) Mutat. Res. 285:125-144; Hayashi (1992) Genet.Anal. Tech. Appl. 9:73-79). The sensitivity of the assay may be enhancedby using RNA (rather than DNA), in which the secondary structure is moresensitive to a change in sequence. In a preferred embodiment, thesubject method utilizes heteroduplex analysis to separatedouble-stranded heteroduplex molecules on the basis of changes inelectrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditions thatpermit hybridization only if a perfect match is found (Saiki et al.(1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci. USA86:6230). Such allele-specific oligonucleotides are hybridized toPCR-amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele-specific amplification technology, which dependson selective PCR amplification, may be used in conjunction with theinstant invention. Oligonucleotides used as primers for specificamplification may carry the mutation of interest in the center of themolecule so that amplification depends on differential hybridization(Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme3′ end of one primer where, under appropriate conditions, mismatch canprevent or reduce polymerase extension (Prossner (1993) Tibtech 11:238).In addition, it may be desirable to introduce a novel restriction sitein the region of the mutation to create cleavage-based detection(Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated thatin certain embodiments amplification may also be performed using Taqligase for amplification (Barany (1991) Proc. Natl. Acad. Sci. USA88:189). In such cases, ligation will occur only if there is a perfectmatch at the 3′ end of the 5′ sequence making it possible to detect thepresence of a known mutation at a specific site by looking for thepresence or absence of amplification.

The methods described herein may be performed, for example, by utilizingprepackaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnosed patients exhibiting symptoms orfamily history of a disease or illness involving an adenylate kinasegene.

3. Pharmacogenomics

Agents or modulators that have a stimulatory or inhibitory effect onadenylate kinase activity (e.g., adenylate kinase gene expression) asidentified by a screening assay described herein, can be administered toindividuals to treat (prophylactically or therapeutically) disordersassociated with aberrant adenylate kinase activity. In conjunction withsuch treatment, the pharmacogenomics (i.e., the study of therelationship between an individual's genotype and that individual'sresponse to a foreign compound or drug) of the individual may beconsidered. Differences in metabolism of therapeutics can lead to severetoxicity or therapeutic failure by altering the relation between doseand blood concentration of the pharmacologically active drug. Thus, thepharmacogenomics of the individual permits the selection of effectiveagents (e.g., drugs) for prophylactic or therapeutic treatments based ona consideration of the individual's genotype. Such pharmacogenomics canfurther be used to determine appropriate dosages and therapeuticregimens. Accordingly, the activity of adenylate kinase protein,expression of adenylate kinase nucleic acid, or mutation content ofadenylate kinase genes in an individual can be determined to therebyselect appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Linder (1997) Clin. Chem.43(2):254-266. In general, two types of pharmacogenetic conditions canbe differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body are referred to as “altered drugaction.” Genetic conditions transmitted as single factors altering theway the body acts on drugs are referred to as “altered drug metabolism”.These pharmacogenetic conditions can occur either as rare defects or aspolymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency(G6PD) is a common inherited enzymopathy in which the main clinicalcomplication is haemolysis after ingestion of oxidant drugs(antimalarials, sulfonamides, analgesics, nitrofurans) and consumptionof fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, a PM will show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the activity of adenylate kinase protein, expression of adenylatekinase nucleic acid, or mutation content of adenylate kinase genes in anindividual can be determined to thereby select appropriate agent(s) fortherapeutic or prophylactic treatment of the individual. In addition,pharmacogenetic studies can be used to apply genotyping of polymorphicalleles encoding drug-metabolizing enzymes to the identification of anindividual's drug responsiveness phenotype. This knowledge, when appliedto dosing or drug selection, can avoid adverse reactions or therapeuticfailure and thus enhance therapeutic or prophylactic efficiency whentreating a subject with an adenylate kinase modulator, such as amodulator identified by one of the exemplary screening assays describedherein.

4. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression or activity of adenylate kinase genes (e.g., the ability tomodulate aberrant cell proliferation and/or differentiation) can beapplied not only in basic drug screening but also in clinical trials.For example, the effectiveness of an agent, as determined by a screeningassay as described herein, to increase or decrease adenylate kinase geneexpression, protein levels, or protein activity, can be monitored inclinical trials of subjects exhibiting decreased or increased adenylatekinase gene expression, protein levels, or protein activity. In suchclinical trials, adenylate kinase expression or activity and preferablythat of other genes that have been implicated in for example, a cellularproliferation disorder, can be used as a marker of the responsiveness ofa particular cell.

For example, and not by way of limitation, genes that are modulated incells by treatment with an agent (e.g., compound, drug, or smallmolecule) that modulates adenylate kinase activity (e.g., as identifiedin a screening assay described herein) can be identified. Thus, to studythe effect of agents on cellular proliferation disorders, for example,in a clinical trial, cells can be isolated and RNA prepared and analyzedfor the levels of expression of adenylate kinase genes and other genesimplicated in the disorder. The levels of gene expression (i.e., a geneexpression pattern) can be quantified by Northern blot analysis orRT-PCR, as described herein, or alternatively by measuring the amount ofprotein produced, by one of the methods as described herein, or bymeasuring the levels of activity of adenylate kinase genes or othergenes. In this way, the gene expression pattern can serve as a marker,indicative of the physiological response of the cells to the agent.Accordingly, this response state may be determined before, and atvarious points during, treatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (1) obtaininga preadministration sample from a subject prior to administration of theagent; (2) detecting the level of expression of an adenylate kinaseprotein, mRNA, or genomic DNA in the preadministration sample; (3)obtaining one or more postadministration samples from the subject; (4)detecting the level of expression or activity of the adenylate kinaseprotein, mRNA, or genomic DNA in the postadministration samples; (5)comparing the level of expression or activity of the adenylate kinaseprotein, mRNA, or genomic DNA in the preadministration sample with theadenylate kinase protein, mRNA, or genomic DNA in the postadministrationsample or samples; and (vi) altering the administration of the agent tothe subject accordingly to bring about the desired effect, i.e., forexample, an increase or a decrease in the expression or activity of anadenylate kinase protein.

C. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant adenylate kinaseexpression or activity. Additionally, the compositions of the inventionfind use in the treatment of disorders described herein. Treatment isdefined as the application or administration of a therapeutic agent to apatient, or application or administration of a therapeutic agent to anisolated tissue or cell line from a patient, who has a disease, asymptom of disease or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease, the symptoms of disease or thepredisposition toward disease. “Subject”, as used herein, can refer to amammal, e.g. a human, or to an experimental or animal or disease model.The subject can also be a non-human animal, e.g. a horse, cow, goat, orother domestic animal. A therapeutic agent includes, but is not limitedto, small molecules, peptides, antibodies, ribozymes and antisenseoligonucleotides. Thus, therapies for disorders associated withadenylate kinase expression are encompassed herein.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject a disease or condition associated with an aberrant adenylatekinase expression or activity by administering to the subject an agentthat modulates adenylate kinase expression or at least one adenylatekinase gene activity. Subjects at risk for a disease that is caused, orcontributed to, by aberrant adenylate kinase expression or activity canbe identified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe adenylate kinase aberrancy, such that a disease or disorder isprevented or, alternatively, delayed in its progression. Depending onthe type of adenylate kinase aberrancy, for example, an adenylate kinaseagonist or adenylate kinase antagonist agent can be used for treatingthe subject. The appropriate agent can be determined based on screeningassays described herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulatingadenylate kinase expression or activity for therapeutic purposes. Themodulatory method of the invention involves contacting a cell with anagent that modulates one or more of the activities of adenylate kinaseprotein activity associated with the cell. An agent that modulatesadenylate kinase protein activity can be an agent as described herein,such as a nucleic acid or a protein, a naturally-occurring cognateligand of an adenylate kinase protein, a peptide, an adenylate kinasepeptidomimetic, or other small molecule. In one embodiment, the agentstimulates one or more of the biological activities of adenylate kinaseprotein. Examples of such stimulatory agents include active adenylatekinase protein and a nucleic acid molecule encoding an adenylate kinaseprotein that has been introduced into the cell. In another embodiment,the agent inhibits one or more of the biological activities of adenylatekinase protein. Examples of such inhibitory agents include antisenseadenylate kinase nucleic acid molecules and anti-adenylate kinaseantibodies.

These modulatory methods can be performed in vitro (e.g., by culturingthe cell with the agent) or, alternatively, in vivo (e.g, byadministering the agent to a subject). As such, the present inventionprovides methods of treating an individual afflicted with a disease ordisorder characterized by aberrant expression or activity of anadenylate kinase protein or nucleic acid molecule. In one embodiment,the method involves administering an agent (e.g., an agent identified bya screening assay described herein), or a combination of agents, thatmodulates (e.g., upregulates or downregulates) adenylate kinaseexpression or activity. In another embodiment, the method involvesadministering an adenylate kinase protein or nucleic acid molecule astherapy to compensate for reduced or aberrant adenylate kinaseexpression or activity.

Stimulation of adenylate kinase activity is desirable in situations inwhich an adenylate kinase protein is abnormally downregulated and/or inwhich increased adenylate kinase activity is likely to have a beneficialeffect. Conversely, inhibition of adenylate kinase activity is desirablein situations in which adenylate kinase activity is abnormallyupregulated and/or in which decreased adenylate kinase activity islikely to have a beneficial effect.

This invention may be embodied in many different forms and should not beconstrued as limited to the embodiments set forth herein; rather, theseembodiments are provided so that this disclosure will fully convey theinvention to those skilled in the art. Many modifications and otherembodiments of the invention will come to mind in one skilled in the artto which this invention pertains having the benefit of the teachingspresented in the foregoing description. Although specific terms areemployed, they are used as in the art unless otherwise indicated.

Other Embodiments

In another aspect, the invention features, a method of analyzing aplurality of capture probes. The method can be used, e.g., to analyzegene expression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g., anucleic acid or peptide sequence; contacting the array with a 27802,preferably purified, nucleic acid, preferably purified, polypeptide,preferably purified, or antibody, and thereby evaluating the pluralityof capture probes. Binding, e.g., in the case of a nucleic acid,hybridization with a capture probe at an address of the plurality, isdetected, e.g., by signal generated from a label attached to the 27802nucleic acid, polypeptide, or antibody.

The capture probes can be a set of nucleic acids from a selected sample,e.g., a sample of nucleic acids derived from a control or non-stimulatedtissue or cell.

The method can include contacting the 27802 nucleic acid, polypeptide,or antibody with a first array having a plurality of capture probes anda second array having a different plurality of capture probes. Theresults of each hybridization can be compared, e.g., to analyzedifferences in expression between a first and second sample. The firstplurality of capture probes can be from a control sample, e.g., a wildtype, normal, or non-diseased, non-stimulated, sample, e.g., abiological fluid, tissue, or cell sample. The second plurality ofcapture probes can be from an experimental sample, e.g., a mutant type,at risk, disease-state or disorder-state, or stimulated, sample, e.g., abiological fluid, tissue, or cell sample.

The plurality of capture probes can be a plurality of nucleic acidprobes each of which specifically hybridizes, with an allele of 27802.Such methods can be used to diagnose a subject, e.g., to evaluate riskfor a disease or disorder, to evaluate suitability of a selectedtreatment for a subject, to evaluate whether a subject has a disease ordisorder. 27802 is associated with adenylate kinase activity, thus it isuseful for disorders associated with abnormal cellular growth and/ormetabolism.

The method can be used to detect SNPs, as described above.

In another aspect, the invention features, a method of analyzing aplurality of probes. The method is useful, e.g., for analyzing geneexpression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the pluralityhaving a unique capture probe, e.g., wherein the capture probes are froma cell or subject which express or misexpress 27802 or from a cell orsubject in which a 27802 mediated response has been elicited, e.g., bycontact of the cell with 27802 nucleic acid or protein, oradministration to the cell or subject 27802 nucleic acid or protein;contacting the array with one or more inquiry probe, wherein an inquiryprobe can be a nucleic acid, polypeptide, or antibody (which ispreferably other than 27802 nucleic acid, polypeptide, or antibody);providing a two dimensional array having a plurality of addresses, eachaddress of the plurality being positionally distinguishable from eachother address of the plurality, and each address of the plurality havinga unique capture probe, e.g., wherein the capture probes are from a cellor subject which does not express 27802 (or does not express as highlyas in the case of the 27802 positive plurality of capture probes) orfrom a cell or subject which in which a 27802 mediated response has notbeen elicited (or has been elicited to a lesser extent than in the firstsample); contacting the array with one or more inquiry probes (which ispreferably other than a 27802 nucleic acid, polypeptide, or antibody),and thereby evaluating the plurality of capture probes. Binding, e.g.,in the case of a nucleic acid, hybridization with a capture probe at anaddress of the plurality, is detected, e.g., by signal generated from alabel attached to the nucleic acid, polypeptide, or antibody.

In another aspect, the invention features, a method of analyzing 27802,e.g., analyzing structure, function, or relatedness to other nucleicacid or amino acid sequences. The method includes: providing a 27802nucleic acid or amino acid sequence; comparing the 27802 sequence withone or more preferably a plurality of sequences from a collection ofsequences, e.g., a nucleic acid or protein sequence database; to therebyanalyze 27802.

Preferred databases include GenBank™. The method can include evaluatingthe sequence identity between a 27802 sequence and a database sequence.The method can be performed by accessing the database at a second site,e.g., over the internet.

In another aspect, the invention features, a set of oligonucleotides,useful, e.g., for identifying SNP's, or identifying specific alleles of27802. The set includes a plurality of oligonucleotides, each of whichhas a different nucleotide at an interrogation position, e.g., an SNP orthe site of a mutation. In a preferred embodiment, the oligonucleotidesof the plurality are identical in sequence with one another (except fordifferences in length). The oligonucleotides can be provided withdifferent labels, such that an oligonucleotide that hybridizes to oneallele provides a signal that is distinguishable from an oligonucleotidewhich hybridizes to a second allele.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

EXPERIMENTAL Example 1 Identification and Characterization of Human27802 cDNAs

The human 27802 sequence (FIG. 28; SEQ ID NO:21), which is approximately1452 nucleotides long including untranslated regions, contains apredicted methionine-initiated coding sequence of about 774 nucleotides(nucleotides 219-992 of SEQ ID NO:21; SEQ ID NO:23). The coding sequenceencodes a 258 amino acid protein (SEQ ID NO:22).

Example 2 Tissue Distribution of 27802 mRNA

Northern blot hybridizations with various RNA samples are performedunder standard conditions and washed under stringent conditions, i.e.,0.2×SSC at 65° C. A DNA probe corresponding to all or a portion of the27802 cDNA (SEQ ID NO:21) can be used. The DNA is radioactively labeledwith ³²P-dCTP using the Prime-It Kit (Stratagene, La Jolla, Calif.)according to the instructions of the supplier. Filters containing mRNAfrom mouse hematopoietic and endocrine tissues, and cancer cell lines(Clontech, Palo Alto, Calif.) are probed in ExpressHyb hybridizationsolution (Clontech) and washed at high stringency according tomanufacturer's recommendations.

Expression levels were determined by quantitative PCR (Taqman® brandquantitative PCR kit, Applied Biosystems). The quantitative PCRreactions were performed according to the kit manufacturer'sinstructions. The highest levels of expression of 27802 were observed inartery, kidney, brain cortex and brain hypothalamus, ovary, lung(tumor), and tonsil (see FIG. 35).

Example 3 Recombinant Expression of 27802 in Bacterial Cells

In this example, 27802 polypeptide is expressed as a recombinantglutathione-S-transferase (GST) fusion polypeptide in E. coli and thefusion polypeptide is isolated and characterized. Specifically, 27802 isfused to GST and this fusion polypeptide is expressed in E. coli, e.g.,strain PEB199. Expression of the GST-27802 fusion protein in PEB199 isinduced with IPTG. The recombinant fusion polypeptide is purified fromcrude bacterial lysates of the induced PEB199 strain by affinitychromatography on glutathione beads. Using polyacrylamide gelelectrophoretic analysis of the polypeptide purified from the bacteriallysates, the molecular weight of the resultant fusion polypeptide isdetermined.

Example 4 Expression of Recombinant 27802 Protein in COS Cells

To express the 27802 gene in COS cells, the pcDNA/Amp vector byInvitrogen Corporation (San Diego, Calif.) is used. This vector containsan SV40 origin of replication, an ampicillin resistance gene, an E. colireplication origin, a CMV promoter followed by a polylinker region, andan SV40 intron and polyadenylation site. A DNA fragment encoding theentire 27802 protein and an HA tag (Wilson et al. (1984) Cell 37:767) ora FLAG tag fused in-frame to its 3′ end of the fragment is cloned intothe polylinker region of the vector, thereby placing the expression ofthe recombinant protein under the control of the CMV promoter.

To construct the plasmid, the 27802 DNA sequence is amplified by PCRusing two primers. The 5′ primer contains the restriction site ofinterest followed by approximately twenty nucleotides of the 27802coding sequence starting from the initiation codon; the 3′ end sequencecontains complementary sequences to the other restriction site ofinterest, a translation stop codon, the HA tag or FLAG tag and the last20 nucleotides of the 27802 coding sequence. The PCR amplified fragmentand the pCDNA/Amp vector are digested with the appropriate restrictionenzymes and the vector is dephosphorylated using the CIAP enzyme (NewEngland Biolabs, Beverly, Mass.). Preferably the two restriction siteschosen are different so that the 27802 gene is inserted in the correctorientation. The ligation mixture is transformed into E. coli cells(strains HB101, DH5α, SURE, available from Stratagene Cloning Systems,La Jolla, Calif., can be used), the transformed culture is plated onampicillin media plates, and resistant colonies are selected. PlasmidDNA is isolated from transformants and examined by restriction analysisfor the presence of the correct fragment.

COS cells are subsequently transfected with the 27802-pcDNA/Amp plasmidDNA using the calcium phosphate or calcium chloride co-precipitationmethods, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Other suitable methods for transfecting host cells canbe found in Sambrook, J., Fritsh, E. F., and Maniatis, T. MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Theexpression of the 27802 polypeptide is detected by radiolabelling(³⁵S-methionine or ³⁵S-cysteine available from NEN, Boston, Mass., canbe used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1988) using an HA specific monoclonal antibody. Briefly,the cells are labeled for 8 hours with ³⁵S-methionine (or ³⁵S-cysteine).The culture media are then collected and the cells are lysed usingdetergents (RIPA buffer, 150 mM NaCl, 1% NP-40,0.1% SDS, 0.5% DOC, 50 mMTris, pH 7.5). Both the cell lysate and the culture media areprecipitated with an HA specific monoclonal antibody. Precipitatedpolypeptides are then analyzed by SDS-PAGE.

Alternatively, DNA containing the 27802 coding sequence is cloneddirectly into the polylinker of the pCDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of the 27802polypeptide is detected by radiolabelling and immunoprecipitation usinga 27802 specific monoclonal antibody.

1. An isolated nucleic acid molecule selected from the group consistingof: a) a nucleic acid molecule comprising a nucleotide sequence which isat least 60% identical to the nucleotide sequence of SEQ ID NO:1, 3, 5,7, 10, 12, 16, 18, 21, or 23, or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession No. ______; b) anucleic acid molecule comprising a fragment of at least 300 nucleotidesof the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 10, 12, 16, 18, 21,or 23, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession No. ______; c) a nucleic acid moleculewhich encodes a polypeptide comprising the amino acid sequence of SEQ IDNO:2, 6, 11, 17 or 22, or the amino acid sequence encoded by the cDNAinsert of the plasmid deposited with the ATCC as Accession No. ______;d) a nucleic acid molecule which encodes a fragment of a polypeptidecomprising the amino acid sequence of SEQ ID NO:2, 6, 11, 17, 22, or theamino acid sequence encoded by the cDNA insert of the plasmid depositedwith the ATCC as Accession No. ______, wherein the fragment comprises atleast 15 contiguous amino acids of SEQ ID NO:2, 6, 11, 17, 22, or theamino acid sequence encoded by the cDNA insert of the plasmid depositedwith the ATCC as Accession Number ______; and e) a nucleic acid moleculewhich encodes a naturally occurring allelic variant of a polypeptidecomprising the amino acid sequence of SEQ ID NO:2, 6, 11, 17, 22, or theamino acid sequence encoded by the cDNA insert of the plasmid depositedwith the ATCC as Accession No. ______, wherein the nucleic acid moleculehybridizes to a nucleic acid molecule comprising the complement of SEQID NO:1, 3, 5, 7, 10, 12, 16, 18, 21, or 23 under stringent conditions.2. The isolated nucleic acid molecule of claim 1, which is selected fromthe group consisting of: a) a nucleic acid comprising the nucleotidesequence of SEQ ID NO: 1, 3, 5, 7, 10, 12, 16, 18, 21, or 23, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession No. ______; and b) a nucleic acid molecule which encodes apolypeptide comprising the amino acid sequence of SEQ ID NO:2, 6, 11,17, or 22, or the amino acid sequence encoded by the cDNA insert of theplasmid deposited with the ATCC as Accession No. ______.
 3. The nucleicacid molecule of claim 1 further comprising vector nucleic acidsequences.
 4. The nucleic acid molecule of claim 1 further comprisingnucleic acid sequences encoding a heterologous polypeptide.
 5. A hostcell which contains the nucleic acid molecule of claim
 1. 6. The hostcell of claim 5 which is a mammalian host cell.
 7. A non-human mammalianhost cell containing the nucleic acid molecule of claim
 1. 8. Anisolated polypeptide selected from the group consisting of: a) apolypeptide which is encoded by a nucleic acid molecule comprising anucleotide sequence which is at least 60% identical to a nucleic acidcomprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 10, 12, 16,18, 21, or 23, or the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession No. ______, or a complementthereof; b) a naturally occurring allelic variant of a polypeptidecomprising the amino acid sequence of SEQ ID NO:2, 6, 11, 17, or 22, orthe amino acid sequence encoded by the cDNA insert of the plasmiddeposited with the ATCC as Accession No. ______, wherein the polypeptideis encoded by a nucleic acid molecule which hybridizes to a nucleic acidmolecule comprising SEQ ID NO: 1, 3, 5, 7, 10, 12, 16, 18, 21, or 23, ora complement thereof under stringent conditions; and c) a fragment of apolypeptide comprising the amino acid sequence of SEQ ID NO:2, 6, 11,17, or 22, or the amino acid sequence encoded by the cDNA insert of theplasmid deposited with the ATCC as Accession No. ______, wherein thefragment comprises at least 15 contiguous amino acids of SEQ ID NO:2, 6,11, 17, or
 22. 9. The isolated polypeptide of claim 8 comprising theamino acid sequence of SEQ ID NO:2, 6, 11, 17,
 22. 10. The polypeptideof claim 8 further comprising heterologous amino acid sequences.
 11. Anantibody which selectively binds to a polypeptide of claim
 8. 12. Amethod for producing a polypeptide selected from the group consistingof: a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2,6, 11, 17, or 22, or the amino acid sequence encoded by the cDNA insertof the plasmid deposited with the ATCC as Accession No. ______; b) apolypeptide comprising a fragment of the amino acid sequence of SEQ IDNO:2, 6, 11, 17, or 22, or the amino acid sequence encoded by the cDNAinsert of the plasmid deposited with the ATCC as Accession No. ______,wherein the fragment comprises at least 15 contiguous amino acids of SEQID NO:2, 6, 11, 17, or 22, or the amino acid sequence encoded by thecDNA insert of the plasmid deposited with the ATCC as Accession No.______; and c) a naturally occurring allelic variant of a polypeptidecomprising the amino acid sequence of SEQ ID NO:2, 6, 11, 17, or 22, orthe amino acid sequence encoded by the cDNA insert of the plasmiddeposited with the ATCC as Accession No. ______, wherein the polypeptideis encoded by a nucleic acid molecule which hybridizes to a nucleic acidmolecule comprising SEQ ID NO: 1, 3, 5, 7, 10, 12, 16, 18, 21, or 23;comprising culturing the host cell of claim 5 under conditions in whichthe nucleic acid molecule is expressed.
 13. A method for detecting thepresence of a polypeptide of claim 8 in a sample, comprising: a)contacting the sample with a compound which selectively binds to apolypeptide of claim 8; and b) determining whether the compound binds tothe polypeptide in the sample.
 14. The method of claim 13, wherein thecompound which binds to the polypeptide is an antibody.
 15. A kitcomprising a compound which selectively binds to a polypeptide of claim8 and instructions for use.
 16. A method for detecting the presence of anucleic acid molecule of claim 1 in a sample, comprising the steps of:a) contacting the sample with a nucleic acid probe or primer whichselectively hybridizes to the nucleic acid molecule; and b) determiningwhether the nucleic acid probe or primer binds to a nucleic acidmolecule in the sample.
 17. The method of claim 16, wherein the samplecomprises mRNA molecules and is contacted with a nucleic acid probe. 18.A kit comprising a compound which selectively hybridizes to a nucleicacid molecule of claim 1 and instructions for use.
 19. A method foridentifying a compound which binds to a polypeptide of claim 8comprising the steps of: a) contacting a polypeptide, or a cellexpressing a polypeptide of claim 8 with a test compound; and b)determining whether the polypeptide binds to the test compound.
 20. Themethod of claim 19, wherein the binding of the test compound to thepolypeptide is detected by a method selected from the group consistingof: a) detection of binding by direct detecting of testcompound/polypeptide binding; and, b) detection of binding using acompetition binding assay.
 21. A method for modulating the activity of apolypeptide of claim 8 comprising contacting a polypeptide or a cellexpressing a polypeptide of claim 8 with a compound which binds to thepolypeptide in a sufficient concentration to modulate the activity ofthe polypeptide.
 22. A method for identifying a compound which modulatesthe activity of a polypeptide of claim 8, comprising: a) contacting apolypeptide of claim 8 with a test compound; and b) determining theeffect of the test compound on the activity of the polypeptide tothereby identify a compound which modulates the activity of thepolypeptide.